Modified amino acids comprising tetrazine functional groups, methods of preparation, and methods of their use

ABSTRACT

Provided herein are modified amino acids comprising a tetrazine groups according to Formula I: polypeptides, antibodies, payloads and conjugates comprising these modified amino acid residues derived from the modified amino acids, and methods of producing the polypeptides, antibodies, payloads and conjugates comprising the modified amino acid residues. The polypeptides, antibodies, payloads and conjugates are useful in methods of treatment and prevention, methods of detection and methods of diagnosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/890,118, filed Oct. 11, 2013, the contents of whichare hereby incorporated by reference in their entirety.

FIELD

Provided herein are modified amino acids comprising a tetrazinefunctional group, polypeptides, antibodies, payloads and conjugatescomprising modified amino acid residues derived from the modified aminoacids, and methods of producing the polypeptides, antibodies, payloadsand conjugates comprising the modified amino acid residues. Thepolypeptides, antibodies, payloads and conjugates are useful in methodsof treatment and prevention, methods of detection and methods ofdiagnosis.

BACKGROUND

Engineered polypeptides are used widely in therapeutic and diagnosticapplications. Therapeutic antibodies have been used for many years in,for example, treatment of cancer and inflammatory conditions.Therapeutic polypeptides are also used to treat and prevent bloodconditions and viral infections. Diagnostic polypeptides have been usedsuccessfully to identify healthy and diseased cells and tissues in vivo.

Many polypeptides can provide targeting functionality to specific cells.The selective affinity of certain polypeptides can be used to targetnearly any cell or tissue desired, for example a cell expressing anantigen. A polypeptide can carry a payload to destroy the target cell ortissue, or to slow its growth. Polypeptides have thus found use intherapy for conditions such as cancer, inflammatory diseases, autoimmunediseases and transplant rejection.

In certain applications therapeutic polypeptides are linked to molecularshields (e.g., macromolecules) to increase their half-life within anorganism. For example, the attachment of polyethylene glycol (PEG) to apolypeptide can render the polypeptide non-detectable or less detectableby a patient's immune system, thereby reducing immunogenicity.

Polypeptides have also found use as diagnostics. These polypeptides cancarry a label whose detection indicates the presence of a targetreceptor or antigen on a cell or in a tissue. These labels are typicallylinked to the polypeptides by covalent bonds.

To date, techniques for linking polypeptides to payloads such asmolecular shields, labels, diagnostic compounds, and therapeuticcompounds have been limited by their heterogeneity in degree andlocation of linking to the polypeptides, by their low yields and bylosses in activity. These problems are particularly acute whenattempting to conjugate more than one payload to a single polypeptide ina controlled manner, to produce a homogeneous product. Typicalconjugation sites include random locations on polypeptide chains, e.g.random amines on amino acid residue side chains, and the N-terminus ofcertain polypeptide chains. In such cases, some polypeptides might belinked to a payload at one location while some polypeptides are linkedto the same payload at another location, and some polypeptides might notbe linked at all. If more than one payload is used, some polypeptidesmay be linked to a single payload, some polypeptides may be linked toall payloads, and some polypeptides may be linked to fewer than allpayloads.

There is a need, therefore, for polypeptides modified at site-specificpositions optimized for uniformity, yield and/or activity to further thepromising use of polypeptides in, for example, therapy and diagnostics.

SUMMARY

Provided herein are modified amino acids comprising a tetrazine group,polypeptides, antibodies, payloads and conjugates comprising themodified amino acid residues derived from the modified amino acids, andmethods of producing the polypeptides, antibodies, payloads andconjugates comprising modified amino acid residues. The polypeptides,antibodies, payloads and conjugates are useful in methods of treatmentand prevention, methods of detection and methods of diagnosis.

In one aspect a compound according to formula I is provided:

or a salt thereof, wherein:

Ar is

V is a single bond, lower alkylene, or —W₁—W₂—; one of W₁ and W₂ isabsent or lower alkylene, and the other is —NH—, —O—, or —S—; each X₁ isindependently —NH—, —O—, or —S—; one of Z₁, Z₂, and Z₃ is —CH— or —N—and the others of Z₁, Z₂, and Z₃ are each independently —CH—; and R islower alkyl. In certain embodiments, when Ar is

and V is —NH—, then one of Z₁, Z₂, and Z₃ is —N—.

In a further aspect, polypeptides and antibodies comprising an aminoacid residue corresponding to a compound of formula I are provided. Inparticular embodiments, conjugates of the polypeptides and payloads areprovided. In further embodiments, conjugates of the antibodies andpayloads are provided.

In another aspect, an orthogonal tRNA is provided aminoacylated with anamino acid residue corresponding to a compound of formula I. In arelated aspect, a method of producing a polypeptide is provided,comprising contacting a polypeptide with an orthogonal tRNAaminoacylated with an amino acid residue corresponding to a compound offormula I.

The compounds of formula I described herein can be incorporated into anypolypeptide known to those of skill in the art. Such polypeptidesinclude, but are not limited to, proteins, antibodies, antibodyfragments, and enzymes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides results demonstrating that tRNA synthetase 2A2incorporates a compound of formula 9 into a GFP polypeptide, asdescribed in Example 4. In FIG. 1, the y-axis shows fluorescence, inrelative fluorescence units (RFU); “aaRS” is 2A2; “GFP” is native GFP(without K49TAG); and “negative control” is a cell-free proteinsynthesis reaction without a DNA template.

FIG. 2 provides results demonstrating that tRNA synthetase 2A9incorporates a compound of formula 6 into a GFP polypeptide, asdescribed in Example 4. In FIG. 2, the y-axis shows fluorescence, inrelative fluorescence units (RFU); “aaRS” is 2A9; “GFP” is native GFP(without K49TAG); and “negative control” is a cell-free proteinsynthesis reaction without a DNA template.

FIG. 3 provides an autoradiogram of an antibody incorporating para-azidomethyl phenylalanine (pAMF) at position S7 of the light chain and atetrazine-containing non-natural amino acid (AB 4091) at position F404of the heavy chain.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Provided herein are compounds of formula I, polypeptides, antibodies,payloads and conjugates comprising the amino acid residues correspondingto the compounds of formula I, and methods of producing thepolypeptides, antibodies, payloads and conjugates comprising themodified amino acid residues corresponding to the compounds of formulaI.

Definitions

When referring to the compounds provided herein, the following termshave the following meanings unless indicated otherwise. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as is commonly understood by one of ordinary skill in the art.In the event that there is a plurality of definitions for a term herein,those in this section prevail unless stated otherwise.

The term “alkyl,” as used herein, unless otherwise specified, refers toa saturated straight or branched hydrocarbon. In certain embodiments,the alkyl group is a primary, secondary, or tertiary hydrocarbon. Incertain embodiments, the alkyl group includes one to ten carbon atoms,i.e., C₁ to C₁₀ alkyl. In certain embodiments, the alkyl group isselected from the group consisting of methyl, CF₃, CCl₃, CFCl₂, CF₂Cl,ethyl, CH₂CF₃, CF₂CF₃, propyl, isopropyl, butyl, isobutyl, secbutyl,t-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl,2,2-dimethylbutyl, and 2,1-dimethylbutyl. The term includes bothsubstituted and unsubstituted alkyl groups, including halogenated alkylgroups. In certain embodiments, the alkyl group is a fluorinated alkylgroup. Non-limiting examples of moieties with which the alkyl group canbe substituted are selected from the group consisting of halogen(fluoro, chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino,alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid,sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected,or protected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons, Second Edition, 1991, herebyincorporated by reference.

The term “lower alkyl,” as used herein, and unless otherwise specified,refers to a saturated straight or branched hydrocarbon having one to sixcarbon atoms, i.e., C₁ to C₆ alkyl. In certain embodiments, the loweralkyl group is a primary, secondary, or tertiary hydrocarbon. The termincludes both substituted and unsubstituted moieties.

The term “cycloalkyl,” as used herein, unless otherwise specified,refers to a saturated cyclic hydrocarbon. In certain embodiments, thecycloalkyl group may be a saturated, and/or bridged, and/or non-bridged,and/or a fused bicyclic group. In certain embodiments, the cycloalkylgroup includes three to ten carbon atoms, i.e., C₃ to C₁₀ cycloalkyl. Insome embodiments, the cycloalkyl has from 3 to 15 (C₃₋₁₅), from 3 to 10(C₃₋₁₀), or from 3 to 7 (C₃₋₇) carbon atoms. In certain embodiments, thecycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cyclohexylmethyl, cycloheptyl, bicyclo[2.1.1]hexyl,bicyclo[2.2.1]heptyl, decalinyl or adamantyl. The term includes bothsubstituted and unsubstituted cycloalkyl groups, including halogenatedcycloalkyl groups. In certain embodiments, the cycloalkyl group is afluorinated cycloalkyl group. Non-limiting examples of moieties withwhich the cycloalkyl group can be substituted are selected from thegroup consisting of halogen (fluoro, chloro, bromo or iodo), hydroxyl,carbonyl, sulfanyl, amino, alkylamino, arylamino, alkoxy, aryloxy,nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, orphosphonate, either unprotected, or protected as necessary.

“Alkylene” refers to divalent saturated aliphatic hydrocarbon groupsparticularly having from one to eleven carbon atoms which can bestraight-chained or branched. In certain embodiments, the alkylene groupcontains 1 to 10 carbon atoms. The term includes both substituted andunsubstituted moieties. This term is exemplified by groups such asmethylene (—CH₂—), ethylene (—CH₂CH₂—), the propylene isomers (e.g.,—CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like. The term includeshalogenated alkylene groups. In certain embodiments, the alkylene groupis a fluorinated alkylene group. Non-limiting examples of moieties withwhich the alkylene group can be substituted are selected from the groupconsisting of halogen (fluoro, chloro, bromo or iodo), hydroxyl,carbonyl, sulfanyl, amino, alkylamino, alkylaryl, arylamino, alkoxy,aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid,phosphate, and phosphonate, either unprotected, or protected asnecessary.

“Alkenyl” refers to monovalent olefinically unsaturated hydrocarbongroups, in certain embodiment, having up to about 11 carbon atoms, from2 to 8 carbon atoms, or from 2 to 6 carbon atoms, which can bestraight-chained or branched and having at least 1 or from 1 to 2 sitesof olefinic unsaturation. The term includes both substituted andunsubstituted moieties. Exemplary alkenyl groups include ethenyl (i.e.,vinyl, or —CH═CH₂), n-propenyl (—CH₂CH═CH₂), isopropenyl (—C(CH₃)═CH₂),and the like. The term includes halogenated alkenyl groups. In certainembodiments, the alkenyl group is a fluorinated alkenyl group.Non-limiting examples of moieties with which the alkenyl group can besubstituted are selected from the group consisting of halogen (fluoro,chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino, alkylamino,arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,phosphonic acid, phosphate, or phosphonate, either unprotected, orprotected as necessary.

The term “cycloalkenyl,” as used herein, unless otherwise specified,refers to an unsaturated cyclic hydrocarbon. In certain embodiments,cycloalkenyl refers to mono- or multicyclic ring systems that include atleast one double bond. In certain embodiments, the cycloalkenyl groupmay be a bridged, non-bridged, and/or a fused bicyclic group. In certainembodiments, the cycloalkyl group includes three to ten carbon atoms,i.e., C₃ to C₁₀ cycloalkyl. In some embodiments, the cycloalkenyl hasfrom 3 to 7 (C₃₋₁₀), or from 4 to 7 (C₃₋₇) carbon atoms. The termincludes both substituted and unsubstituted cycloalkenyl groups,including halogenated cycloalkenyl groups. In certain embodiments, thecycloalkenyl group is a fluorinated cycloalkenyl group. Non-limitingexamples of moieties with which the cycloalkenyl group can besubstituted are selected from the group consisting of halogen (fluoro,chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino, alkylamino,arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,phosphonic acid, phosphate, or phosphonate, either unprotected, orprotected as necessary.

“Alkenylene” refers to divalent olefinically unsaturated hydrocarbongroups, in certain embodiments, having up to about 11 carbon atoms orfrom 2 to 6 carbon atoms which can be straight-chained or branched andhaving at least 1 or from 1 to 2 sites of olefinic unsaturation. Thisterm is exemplified by groups such as ethenylene (—CH═CH—), thepropenylene isomers (e.g., —CH═CHCH₂— and —C(CH₃)═CH— and —CH═C(CH₃)—)and the like. The term includes both substituted and unsubstitutedalkenylene groups, including halogenated alkenylene groups. In certainembodiments, the alkenylene group is a fluorinated alkenylene group.Non-limiting examples of moieties with which the alkenylene group can besubstituted are selected from the group consisting of halogen (fluoro,chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino, alkylamino,arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,phosphonic acid, phosphate, or phosphonate, either unprotected, orprotected as necessary.

“Alkynyl” refers to acetylenically unsaturated hydrocarbon groups, incertain embodiments, having up to about 11 carbon atoms or from 2 to 6carbon atoms which can be straight-chained or branched and having atleast 1 or from 1 to 2 sites of alkynyl unsaturation. Non-limitingexamples of alkynyl groups include acetylenic, ethynyl (—C≡CH),propargyl (—CH₂C≡CH), and the like. The term includes both substitutedand unsubstituted alkynyl groups, including halogenated alkynyl groups.In certain embodiments, the alkynyl group is a fluorinated alkynylgroup. Non-limiting examples of moieties with which the alkynyl groupcan be substituted are selected from the group consisting of halogen(fluoro, chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino,alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid,sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected,or protected as necessary.

The term “aryl,” as used herein, and unless otherwise specified, refersto phenyl, biphenyl or naphthyl. The term includes both substituted andunsubstituted moieties. An aryl group can be substituted with anydescribed moiety, including, but not limited to, one or more moietiesselected from the group consisting of halogen (fluoro, chloro, bromo oriodo), alkyl, haloalkyl, hydroxyl, amino, alkylamino, arylamino, alkoxy,aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid,phosphate, or phosphonate, either unprotected, or protected asnecessary, as known to those skilled in the art, for example, as taughtin Greene, et al., Protective Groups in Organic Synthesis, John Wileyand Sons, Second Edition, 1991.

“Alkoxy” refers to the group —OR′ where R′ is alkyl or cycloalkyl.Alkoxy groups include, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy,1,2-dimethylbutoxy, and the like.

“Alkoxycarbonyl” refers to a radical —C(O)-alkoxy where alkoxy is asdefined herein.

“Amino” refers to the radical —NH₂.

“Carboxyl” or “carboxy” refers to the radical —C(O)OH.

The term “alkylamino” or “arylamino” refers to an amino group that hasone or two alkyl or aryl substituents, respectively. In certainembodiments, the alkyl substituent is lower alkyl. In some embodiments,the alkyl or lower alkyl is unsubstituted.

“Halogen” or “halo” refers to chloro, bromo, fluoro or iodo.

“Thioalkoxy” refers to the group —SR′ where R′ is alkyl or cycloalkyl.

The term “heterocyclyl” or “heterocyclic” refers to a monovalentmonocyclic non-aromatic ring system and/or multicyclic ring system thatcontains at least one non-aromatic ring, wherein one or more of thenon-aromatic ring atoms are heteroatoms independently selected from O,S, or N; and the remaining ring atoms are carbon atoms. In certainembodiments, the heterocyclyl or heterocyclic group has from 3 to 20,from 3 to 15, from 3 to 10, from 3 to 8, from 4 to 7, or from 5 to 6ring atoms. Heterocyclyl groups are bonded to the rest of the moleculethrough the non-aromatic ring. In certain embodiments, the heterocyclylis a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, whichmay include a fused or bridged ring system, and in which the nitrogen orsulfur atoms may be optionally oxidized, the nitrogen atoms may beoptionally quaternized, and some rings may be partially or fullysaturated, or aromatic. The heterocyclyl may be attached to the mainstructure at any heteroatom or carbon atom which results in the creationof a stable compound. Examples of such heterocyclic radicals include,but are not limited to, azepinyl, benzodioxanyl, benzodioxolyl,benzopyranonyl, benzopyranonyl, benzopyranyl, benzotetrahydrofuranyl,benzotetrahydrothienyl, benzothiopyranyl, benzoxazinyl, β-carbolinyl,chromanyl, chromonyl, cinnolinyl, coumarinyl, decahydroisoquinolinyl,dihydrobenzisothiazinyl, dihydrobenzisoxazinyl, dihydrofuryl,dihydroisoindolyl, dihydropyranyl, dihydropyrazolyl, dihydropyrazinyl,dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl,1,4-dithianyl, furanonyl, imidazolidinyl, imidazolinyl, indolinyl,isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isochromanyl,isocoumarinyl, isoindolinyl, isothiazolidinyl, isoxazolidinyl,morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazolidinonyl,oxazolidinyl, oxiranyl, piperazinyl, piperidinyl, 4-piperidonyl,pyrazolidinyl, pyrazolinyl, pyrrolidinyl, pyrrolinyl, quinuclidinyl,tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydropyranyl,tetrahydrothienyl, thiamorpholinyl, thiazolidinyl, tetrahydroquinolinyl,and 1,3,5-trithianyl. In certain embodiments, heterocyclic may also beoptionally substituted as described herein.

The term “heteroaryl” refers to refers to a monovalent monocyclicaromatic group and/or multicyclic aromatic group that contain at leastone aromatic ring, wherein at least one aromatic ring contains one ormore heteroatoms independently selected from O, S and N in the ring.Heteroaryl groups are bonded to the rest of the molecule through thearomatic ring. Each ring of a heteroaryl group can contain one or two Oatoms, one or two S atoms, and/or one to four N atoms, provided that thetotal number of heteroatoms in each ring is four or less and each ringcontains at least one carbon atom. In certain embodiments, theheteroaryl has from 5 to 20, from 5 to 15, or from 5 to 10 ring atoms.Examples of monocyclic heteroaryl groups include, but are not limitedto, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl,oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl,pyrimidinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl,triazinyl and triazolyl. Examples of bicyclic heteroaryl groups include,but are not limited to, benzofuranyl, benzimidazolyl, benzoisoxazolyl,benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl,benzotriazolyl, benzoxazolyl, furopyridyl, imidazopyridinyl,imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl,isobenzothienyl, isoindolyl, isoquinolinyl, isothiazolyl,naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl,pyridopyridyl, pyrrolopyridyl, quinolinyl, quinoxalinyl, quinazolinyl,thiadiazolopyrimidyl, and thienopyridyl. Examples of tricyclicheteroaryl groups include, but are not limited to, acridinyl,benzindolyl, carbazolyl, dibenzofuranyl, perimidinyl, phenanthrolinyl,phenanthridinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxazinyland xanthenyl. In certain embodiments, heteroaryl may also be optionallysubstituted as described herein.

The term “alkylaryl” refers to an aryl group with an alkyl substituent.The term “aralkyl” or “arylalkyl” refers to an alkyl group with an arylsubstituent.

The term “protecting group” as used herein and unless otherwise definedrefers to a group that is added to an oxygen, nitrogen or phosphorusatom to prevent its further reaction or for other purposes. A widevariety of oxygen and nitrogen protecting groups are known to thoseskilled in the art of organic synthesis.

“Pharmaceutically acceptable salt” refers to any salt of a compoundprovided herein which retains its biological properties and which is nottoxic or otherwise undesirable for pharmaceutical use. Such salts may bederived from a variety of organic and inorganic counter-ions well knownin the art. Such salts include, but are not limited to: (1) acidaddition salts formed with organic or inorganic acids such ashydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic,acetic, trifluoroacetic, trichloroacetic, propionic, hexanoic,cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic,succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric,benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic,phthalic, lauric, methanesulfonic, ethanesulfonic,1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic,4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic,camphoric, camphorsulfonic,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic,3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric,gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic,cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2) baseaddition salts formed when an acidic proton present in the parentcompound either (a) is replaced by a metal ion, e.g., an alkali metalion, an alkaline earth ion or an aluminum ion, or alkali metal oralkaline earth metal hydroxides, such as sodium, potassium, calcium,magnesium, aluminum, lithium, zinc, and barium hydroxide, ammonia or (b)coordinates with an organic base, such as aliphatic, alicyclic, oraromatic organic amines, such as ammonia, methylamine, dimethylamine,diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine,ethylenediamine, lysine, arginine, ornithine, choline,N,N′-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine,N-benzylphenethylamine, N-methylglucamine piperazine,tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and thelike.

Pharmaceutically acceptable salts further include, by way of exampleonly and without limitation, sodium, potassium, calcium, magnesium,ammonium, tetraalkylammonium and the like, and when the compoundcontains a basic functionality, salts of non-toxic organic or inorganicacids, such as hydrohalides, e.g. hydrochloride and hydrobromide,sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate,trichloroacetate, propionate, hexanoate, cyclopentylpropionate,glycolate, glutarate, pyruvate, lactate, malonate, succinate, sorbate,ascorbate, malate, maleate, fumarate, tartarate, citrate, benzoate,3-(4-hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate, phthalate,laurate, methanesulfonate (mesylale), ethanesulfonate,1,2-ethane-disulfonate, 2-hydroxyethanesulthnate, benzenesulfonate(besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate,4-toluenesulfonate, camphorate, camphorsulfonate,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate,3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfate,gluconate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate,cyclohexylsulfamate, quinate, muconate and the like.

The term “acyl” or “O-linked ester” refers to a group of the formulaC(O)R′, wherein R′ is alkyl or cycloalkyl (including lower alkyl),carboxylate reside of amino acid, aryl including phenyl, alkaryl,arylalkyl including benzyl, alkoxyalkyl including methoxymethyl,aryloxyalkyl such as phenoxymethyl; or substituted alkyl (includinglower alkyl), aryl including phenyl optionally substituted with chloro,bromo, fluoro, iodo, C₁ to C₄ alkyl or C₁ to C₄ alkoxy, sulfonate esterssuch as alkyl or arylalkyl sulphonyl including methanesulfonyl, themono, di or triphosphate ester, trityl or monomethoxy-trityl,substituted benzyl, alkaryl, arylalkyl including benzyl, alkoxyalkylincluding methoxymethyl, aryloxyalkyl such as phenoxymethyl. Aryl groupsin the esters optimally comprise a phenyl group. In particular, acylgroups include acetyl, trifluoroacetyl, methylacetyl, cyclpropylacetyl,propionyl, butyryl, hexanoyl, heptanoyl, octanoyl, neo-heptanoyl,phenylacetyl, 2-acetoxy-2-phenylacetyl, diphenylacetyl,α-methoxy-α-trifluoromethyl-phenylacetyl, bromoacetyl,2-nitro-benzeneacetyl, 4-chloro-benzeneacetyl,2-chloro-2,2-diphenylacetyl, 2-chloro-2-phenylacetyl, trimethylacetyl,chlorodifluoroacetyl, perfluoroacetyl, fluoroacetyl,bromodifluoroacetyl, methoxyacetyl, 2-thiopheneacetyl, chlorosulfonylacetyl, 3-methoxyphenylacetyl, phenoxyacetyl, tert-butylacetyl,trichloroacetyl, monochloro-acetyl, dichloroacetyl,7H-dodecafluoro-heptano yl, perfluoro-heptanoyl,7H-dodeca-fluoroheptanoyl, 7-chlorododecafluoro-heptanoyl,7-chloro-dodecafluoro-heptanoyl, 7H-dodecafluoroheptanoyl,7H-dodeca-fluoroheptanoyl, nona-fluoro-3,6-dioxa-heptanoyl,nonafluoro-3,6-dioxaheptanoyl, perfluoroheptanoyl, methoxybenzoyl,methyl 3-amino-5-phenylthiophene-2-carboxyl,3,6-dichloro-2-methoxy-benzoyl, 4-(1,1,2,2-tetrafluoro-ethoxy)-benzoyl,2-bromo-propionyl, omega-aminocapryl, decanoyl, n-pentadecanoyl,stearyl, 3-cyclopentyl-propionyl, 1-benzene-carboxyl, 0-acetylmandelyl,pivaloyl acetyl, 1-adamantane-carboxyl, cyclohexane-carboxyl,2,6-pyridinedicarboxyl, cyclopropane-carboxyl, cyclobutane-carboxyl,perfluorocyclohexyl carboxyl, 4-methylbenzoyl, chloromethyl isoxazolylcarbonyl, perfluorocyclohexyl carboxyl, crotonyl,1-methyl-1H-indazole-3-carbonyl, 2-propenyl, isovaleryl,1-pyrrolidinecarbonyl, 4-phenylbenzyl.

The term “amino acid” refers to naturally occurring and synthetic α, β γor δ amino acids, and includes but is not limited to, amino acidresidues found in proteins, i.e. glycine, alanine, valine, leucine,isoleucine, methionine, phenylalanine, tryptophan, proline, serine,threonine, cysteine, tyrosine, asparagine, glutamine, aspartate,glutamate, lysine, arginine and histidine. In certain embodiments, theamino acid is in the L-configuration. Alternatively, the amino acid canbe a derivative of alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl,phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl,threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl,glutaroyl, lysinyl, argininyl, histidinyl, β-alanyl, β-valinyl,β-leucinyl, β-isoleuccinyl, β-prolinyl, β-phenylalaninyl,β-tryptophanyl, β-methioninyl, β-glycinyl, β-serinyl, β-threoninyl,β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl, β-aspartoyl,β-glutaroyl, β-lysinyl, β-argininyl or β-histidinyl.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to polymers of naturally occurring amino acid residuesas well as polymers in which one or more amino acid residues is amodified amino acid residue. Additionally, such “polypeptides,”“peptides” and “proteins” include amino acid residue chains of anylength, including full length proteins, wherein the amino acid residuesare linked by covalent peptide bonds.

The term “substantially free of” or “substantially in the absence of”with respect to a chemical composition refers to a chemical compositionthat includes at least 85% or 90% by weight, in certain embodiments 95%,98%, 99% or 100% by weight, of the designated chemical.

Similarly, the term “isolated” with respect to a chemical compositionrefers to a chemical composition that includes at least 85%, 90%, 95%,98%, 99% to 100% by weight, of the chemical, the remainder comprisingother species or enantiomers.

“Solvate” refers to a compound provided herein or a salt thereof, thatfurther includes a stoichiometric or non-stoichiometric amount ofsolvent bound by non-covalent intermolecular forces. Where the solventis water, the solvate is a hydrate.

“Isotopic composition” refers to the amount of each isotope present fora given atom, and “natural isotopic composition” refers to the naturallyoccurring isotopic composition or abundance for a given atom. Atomscontaining their natural isotopic composition may also be referred toherein as “non-enriched” atoms. Unless otherwise designated, the atomsof the compounds recited herein are meant to represent any stableisotope of that atom. For example, unless otherwise stated, when aposition is designated specifically as “H” or “hydrogen,” the positionis understood to have hydrogen at its natural isotopic composition.

“Isotopic enrichment” refers to the percentage of incorporation of anamount of a specific isotope at a given atom in a molecule in the placeof that atom's natural isotopic abundance. For example, deuteriumenrichment of 1% at a given position means that 1% of the molecules in agiven sample contain deuterium at the specified position. Because thenaturally occurring distribution of deuterium is about 0.0156%,deuterium enrichment at any position in a compound synthesized usingnon-enriched starting materials is about 0.0156%. The isotopicenrichment of the compounds provided herein can be determined usingconventional analytical methods known to one of ordinary skill in theart, including mass spectrometry and nuclear magnetic resonancespectroscopy.

“Isotopically enriched” refers to an atom having an isotopic compositionother than the natural isotopic composition of that atom. “Isotopicallyenriched” may also refer to a compound containing at least one atomhaving an isotopic composition other than the natural isotopiccomposition of that atom.

As used herein, “alkyl,” “cycloalkyl,” “alkenyl,” “cycloalkenyl,”“alkynyl,” “aryl,” “alkoxy,” “alkoxycarbonyl,” “amino,” “carboxyl,”“alkylamino,” “arylamino,” “thioalkyoxy,” “heterocyclyl,” “heteroaryl,”“alkylheterocyclyl,” “alkylheteroaryl,” “acyl,” “aralkyl,” “alkaryl,”“purine,” “pyrimidine,” “carboxyl” and “amino acid” groups optionallycomprise deuterium at one or more positions where hydrogen atoms arepresent, and wherein the deuterium composition of the atom or atoms isother than the natural isotopic composition.

Also as used herein, “alkyl,” “cycloalkyl,” “alkenyl,” “cycloalkenyl,”“alkynyl,” “aryl,” “alkoxy,” “alkoxycarbonyl,” “carboxyl,” “alkylamino,”“arylamino,” “thioalkyoxy,” “heterocyclyl,” “heteroaryl,”“alkylheterocyclyl,” “alkylheteroaryl,” “acyl,” “aralkyl,” “alkaryl,”“purine,” “pyrimidine,” “carboxyl” and “amino acid” groups optionallycomprise carbon-13 at an amount other than the natural isotopiccomposition.

As used herein, EC₅₀ refers to a dosage, concentration or amount of aparticular test compound that elicits a dose-dependent response at 50%of maximal expression of a particular response that is induced, provokedor potentiated by the particular test compound.

As used herein, the IC₅₀ refers to an amount, concentration or dosage ofa particular test compound that achieves a 50% inhibition of a maximalresponse in an assay that measures such response.

As used herein, the terms “subject” and “patient” are usedinterchangeably herein. The terms “subject” and “subjects” refer to ananimal, such as a mammal including a non-primate (e.g., a cow, pig,horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey such as acynomolgous monkey, a chimpanzee and a human), and for example, a human.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to any agent(s) which can be used in the treatment or preventionof a disorder or one or more symptoms thereof. In certain embodiments,the term “therapeutic agent” includes a compound provided herein. Incertain embodiments, a therapeutic agent is an agent which is known tobe useful for, or has been or is currently being used for the treatmentor prevention of a disorder or one or more symptoms thereof.

“Therapeutically effective amount” refers to an amount of a compound orcomposition that, when administered to a subject for treating a disease,is sufficient to effect such treatment for the disease. A“therapeutically effective amount” can vary depending on, inter alia,the compound, the disease and its severity, and the age, weight, etc.,of the subject to be treated.

“Treating” or “treatment” of any disease or disorder refers, in certainembodiments, to ameliorating a disease or disorder that exists in asubject. In some embodiments, “treating” or “treatment” includesamelidrating at least one physical parameter, which may be indiscernibleby the subject. In certain embodiments, “treating” or “treatment”includes modulating the disease or disorder, either physically (e.g.,stabilization of a discernible symptom) or physiologically (e.g.,stabilization of a physical parameter) or both. In some embodiments,“treating” or “treatment” includes delaying the onset of the disease ordisorder.

As used herein, the terms “prophylactic agent” and “prophylactic agents”as used refer to any agent(s) which can be used in the prevention of adisorder or one or more symptoms thereof. In certain embodiments, theterm “prophylactic agent” includes a compound provided herein. Incertain other embodiments, the term “prophylactic agent” does not refera compound provided herein. For example, a prophylactic agent is anagent which is known to be useful for, or has been or is currently beingused to prevent or impede the onset, development, progression and/orseverity of a disorder.

As used herein, the phrase “prophylactically effective amount” refers tothe amount of a therapy (e.g., prophylactic agent) which is sufficientto result in the prevention or reduction of the development, recurrenceor onset of one or more symptoms associated with a disorder, or toenhance or improve the prophylactic effect(s) of another therapy (e.g.,another prophylactic agent).

The term “substantially pure” with respect to a composition comprising amodified amino acid residue refers to a composition that includes atleast 80, 85, 90 or 95% by weight or, in certain embodiments, 95, 98, 99or 100% by weight, e.g. dry weight, of the modified amino acid residuerelative to the remaining portion of the composition. The weightpercentage can be relative to the total weight of protein in thecomposition or relative to the total weight of modified amino acidresidues in the composition. Purity can be determined by techniquesapparent to those of skill in the art.

The term “antibody” refers to any macromolecule that would be recognizedas an antibody by those of skill in the art. Antibodies share commonproperties including binding to an antigen and a structure comprising atleast one polypeptide chain with a region or regions substantiallysimilar to a polypeptide encoded by any of the immunoglobulin genesrecognized by those of skill in the art. The immunoglobulin genesinclude, but are not limited to, the κ, λ, α, γ (IgG1, IgG2, IgG3, andIgG4), δ, ε and μ constant region genes, as well as the immunoglobulinvariable region genes (e.g., IGHV, IGHD, IGHJ, IGLV, IGKV, IGLJ, andIGKJ genes). The term includes full-length antibodies and antibodyfragments recognized by those of skill in the art, and variants thereof.

The term “antibody fragment” refers to any form of an antibody otherthan the full-length form. Antibody fragments herein include antibodiesthat are smaller components that exist within full-length antibodies,and antibodies that have been engineered. Antibody fragments include butare not limited to Fv, Fc, Fab, and (Fab)₂, single chain Fv (scFv),domain antibodies (dAbs), diabodies, triabodies, tetrabodies,bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDR's,variable regions, framework regions, constant regions, and the like(Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson,1998, Curr. Opin. Biotechnol. 9:395-402).

The term “immunoglobulin (Ig)” refers to a protein consisting of one ormore polypeptides substantially encoded by one or more of theimmunoglobulin genes, or a protein substantially identical thereto inamino acid residue sequence. Immunoglobulins include but are not limitedto antibodies. Immunoglobulins may have a number of structural forms,including but not limited to full-length antibodies, antibody fragments,and individual immunoglobulin domains including but not limited toV_(H), D_(H), J_(H), C_(H) (e.g., Cγ1, Cγ2, Cγ3), V_(L), J_(L), andC_(L) (e.g., V_(κ) and V_(λ)).

The term “immunoglobulin (Ig) domain” refers to a protein domainconsisting of a polypeptide substantially encoded by an immunoglobulingene. Ig domains include but are not limited to V_(H), D_(H), J_(H),C_(H) (e.g., Cγ1, Cγ2, Cγ3), V_(L), J_(L), and C_(L) (e.g., V_(κ) andV_(λ)).

The term “variable region” of an antibody refers to a polypeptide orpolypeptides composed of the V_(H) immunoglobulin domain, the V_(L)immunoglobulin domain, or the V_(H) and V_(L) immunoglobulin domains.Variable region may refer to this or these polypeptides in isolation, oras a fragment (e.g., as an Fv fragment or as an scFv fragment), as thisregion in the context of a larger antibody fragment, or as this regionin the context of a full-length antibody or an alternative, non-antibodyscaffold molecule.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areresponsible for the binding specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in six segments called complementarity determining regions(CDRs). Three of these CDRs are located in the light chain variabledomain and three in the heavy chain variable domain. The conservedportions of the variable domains are called the framework regions (FR).The variable domains of native heavy and light chains each comprise fourFR regions, largely adopting a β-sheet configuration, connected by threeCDRs, which form loops connecting, and in some cases forming part of,the β-sheet structure. The CDRs in each chain are held together in closeproximity by the FR regions and, with the CDRs from the other chain,contribute to the formation of the antigen binding site of antibodies(see Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991)).

The constant domains are typically not directly involved in the bindingof an antibody to an antigen, but exhibit various effector functions.Depending on the amino acid residue sequence of the constant region ofthe heavy chains, antibodies can be assigned to different classes. Thereare five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM,and several of these may be further divided into subclasses (isotypes),e.g. IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavy chain constantregions that correspond to the different classes of immunoglobulins arealso called α, δ, ε, γ and μ respectively. Of the various humanimmunoglobulin classes, only human IgG1, IgG2, IgG3 and IgM are known toactivate complement.

The term “conjugate” generally refers to a moiety comprising a modifiedamino acid residue, as described herein, attached to a payload. The term“payload” generally refers to a moiety that is attached (or“conjugated”) to a modified amino acid residue, as described herein. Forexample, a conjugate may comprise a polypeptide or an antibodycomprising a modified amino acid residue attached to a payload. Apayload may be a small molecule or a macromolecule. In some embodiments,the payload is a bioactive molecule including, but not limited to, aprotein, a peptide, a nucleic active or a hybrid thereof. In someembodiments, the payload is a polymer such as polyethylene glycol. Insome embodiments, the payload is a therapeutic agent, such as a drug. Insome embodiments, the payload is a label that can recognize and bind tospecific targets, such as a payload that is useful for detection ordiagnosis. In some embodiments, the payload is connected to a modifiedamino acid residue via a linker. In some embodiments, the payload isdirectly connected to a modified amino acid residue without a linker.

The term “variant protein sequence” refers to a protein sequence thathas one or more residues that differ in amino acid residue identity fromanother similar protein sequence. Said similar protein sequence may bethe natural wild type protein sequence, or another variant of the wildtype sequence. Variants include proteins that have one or more aminoacid residue insertions, deletions or substitutions. Variants alsoinclude proteins that have one or more post-translationally modifiedamino acid residues.

The term “parent antibody” refers to an antibody that is modifiedaccording to the description provided herein. The modification can bephysical, i.e., chemically or biochemically replacing or modifying oneor more amino acid residues of the parent antibody to yield an antibodywithin the scope of the present description. The modification can alsobe conceptual, i.e., using the sequence of one or more polypeptidechains of the parent antibody to design an antibody comprising one ormore site-specific modified amino acid residues according to the presentdescription. Parent antibodies can be naturally occurring antibodies orantibodies designed or developed in a laboratory. Parent antibodies canalso be artificial or engineered antibodies, e.g., chimeric or humanizedantibodies.

The term “conservatively modified variant” refers to a protein thatdiffers from a related protein by conservative substitutions in theamino acid residue sequence. One of skill in the art will recognize thatindividual substitutions, deletions or additions to a peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid residue or a small percentage of amino acid residues in theencoded sequence is a “conservatively modified variant” where thealteration results in the substitution of an amino acid residue with achemically similar amino acid residue. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   -   1) Alanine (A), Glycine (G);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   7) Serine (S), Threonine (T); and    -   8) Cysteine (C), Methionine (M).

See, e.g., Creighton, Proteins: Structures and Molecular Properties, W HFreeman & Co.; 2nd edition (December 1993).

The terms “identical” or “identity,” in the context of two or morepolypeptide sequences, refer to two or more sequences or subsequencesthat are the same. Sequences are “substantially identical” if they havea percentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, optionally about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about98%, about 99%, about 99.5%, or about 99.9% identity over a specifiedregion), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. The identity can exist over a region that is at leastabout 50 amino acid residues or nucleotides in length, over a regionthat is about 10-17 residues in length (e.g., the approximate length ofCDRL1), over a region that is about 7 residues in length (e.g., theapproximate length of CDRL2), over a region that is about 7-11 residuesin length (e.g., the approximate length of CDRL3), over a region that isabout 10-12 residues in length (e.g., the approximate length of CDRH1),over a region that is about 16-19 residues in length (e.g., theapproximate length of CDRH2), over a region that is about 3-35 residuesin length (e.g., the approximate length of CDRH3), or over a region thatis 75-100 amino acid residues or nucleotides in length, or, where notspecified, across the entire sequence or a polypeptide. In the case ofantibodies, identity can be measured outside the variable CDRs. Identitycan also be measured within the entirety of the heavy or light chains,or within the variable regions of the heavy or light chains. Optimalalignment of sequences for comparison can be conducted by methodsincluding, but not limited to, the local homology algorithm of Smith andWaterman (1970) Adv. Appl. Math. 2:482c, the homology alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, thesearch for similarity method of Pearson and Lipman (1988) Proc. Nat'l.Acad. Sci. USA 85:2444, computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.); or bymanual alignment and visual inspection (see, e.g., Ausubel et al.,Current Protocols in Molecular Biology (1995 supplement)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity include the BLAST and BLAST2.0 algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information. TheBLAST algorithm parameters W, T, and X determine the sensitivity andspeed of the alignment. The BLASTN program (fui nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5,N=−4 and a comparison of both strands. For amino acid residue sequences,the BLASTP program uses as defaults a wordlength of 3, and expectation(E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff(1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50,expectation (E) of 10, M=5, N=−4, and a comparison of both strands. TheBLAST algorithm is typically performed with the “low complexity” filterturned off. In some embodiments, the BLAST algorithm is typicallyperformed with the “low complexity” filter turned on.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid residue sequences would occur by chance.For example, a nucleic acid is considered similar to a referencesequence if the smallest sum probability in a comparison of the testnucleic acid to the reference nucleic acid is less than about 0.2, morepreferably less than about 0.01, and most preferably less than about0.001.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acids such as proline, aminoacid analogs and amino acid mimetics that function in a manner similarto naturally occurring amino acids.

Naturally encoded amino acids are the proteinogenic amino acids known tothose of skill in the art. They include the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and the less common pyrrolysine andselenocysteine. Naturally encoded amino acids include post-translationalvariants of the 22 naturally occurring amino acids such as prenylatedamino acids, isoprenylated amino acids, myrisoylated amino acids,palmitoylated amino acids, N-linked glycosylated amino acids, O-linkedglycosylated amino acids, phosphorylated amino acids and acylated aminoacids.

The term “modified amino acid” refers to an amino acid that is not aproteinogenic amino acid, or a post-translationally modified variantthereof. In particular, the term refers to an amino acid that is not oneof the 20 common amino acids or pyrrolysine or selenocysteine, orpost-translationally modified variants thereof.

The term “strained alkene” refers to a molecule comprising an alkenemoiety that is capable of reacting with tetrazine in a tetrazineligation. Exemplary tetrazine ligations are described in Blackman etal., 2008, J. Am. Chem. Soc. 130:13518-13519. Examples includetrans-cyclooctenes and norbornenes. Useful compounds include, but arenot limited to, trans-cyclooctene, (E)-cyclooct-4-enol,(E)-cyclooct-4-enyl 2,5-dioxo-1-pyrrolidinyl carbonate,5-norbornene-2-acetic acid succinimidyl ester, and5-norbornene-2-endo-acetic acid.

Compounds

Provided herein are compounds according to formula I:

or a salt thereof, wherein Ar is:

V is a single bond, lower alkylene, or —W₁—W₂—; one of W₁ and W₂ isabsent or lower alkylene, and the other is —NH—, —O—, or —S—; each X₁ isindependently —NH—, —O—, or —S—; one of Z₁, Z₂, and Z₃ is —CH— or —N—and the others of Z₁, Z₂, and Z₃ are each independently —CH—; and R islower alkyl. In certain embodiments, when Ar is

and V is —NH—, then one of Z₁, Z₂, and Z₃ is —N—. In certainembodiments, V is a single bond, —NH—, or —CH₂NH—.

In certain embodiments, Ar is

and Z₁, Z₂, Z₃ and X₁ are as defined in the context of formula I. Incertain embodiments according to this paragraph, V is —W₁—W₂—; one of W₁and W₂ is absent or —CH₂—, and the other is —NH—, —O—, or —S—. Incertain embodiments according to this paragraph, V is a single bond,—NH—, or —CH₂NH—. In certain embodiments according to this paragraph, Z₁is N. In certain embodiments according to this paragraph, Z₂ is N. Incertain embodiments according to this paragraph, Z₃ is N. In certainembodiments according to this paragraph, Z₁ is CH, Z₃ is CH and X₁ is S.

In certain embodiments, Ar is

and Z₁, Z₂, and Z₃ are as defined in the context of formula I. Incertain embodiments according to this paragraph, V is —W₁—W₂—; one of W₁and W₂ is absent or —CH₂—, and the other is —NH—, —O—, or —S—. Incertain embodiments according to this paragraph, V is a single bond,—NH—, or —CH₂NH—. In certain embodiments according to this paragraph, Z₁is N. In certain embodiments according to this paragraph, Z₂ is N. Incertain embodiments according to this paragraph, Z₃ is N.

In certain embodiments, Ar is

and Z₁, Z₃ and X₁ are as defined in the context of formula I. In certainembodiments according to this paragraph, V is —W₁—W₂—; one of W₁ and W₂is absent or —CH₂—, and the other is —NH—, —O—, or —S—. In certainembodiments according to this paragraph, V is a single bond, —NH—, or—CH₂NH—. In certain embodiments according to this paragraph, Z₁ is N. Incertain embodiments according to this paragraph, Z₃ is N. In certainembodiments according to this paragraph, Z₁ is CH, Z₃ is CH and X₁ is S.

In an embodiment, a compound according to formula Ia is provided:

where Ar, V, and R are defined in the context of formula I.

In an embodiment, compounds of either of formulas I and Ia are providedwherein V is a single bond. In another embodiment, compounds of eitherof formulas I and Ia are provided wherein V is —NH—. In anotherembodiment, compounds of either of formulas I and Ia are providedwherein V is —CH₂NH—.

In an embodiment, a compound according to formula II is provided:

or a salt thereof, wherein V and R are as defined in Formula I. Incertain embodiments according to this paragraph, V is —W₁—W₂—; one of W₁and W₂ is absent or —CH₂—, and the other is —NH—, —O—, or —S—. Incertain embodiments, V is a single bond, —NH—, or —CH₂NH—. In certainembodiments, V is a single bond or —CH₂NH—; and R is methyl.

In an embodiment, a compound according to formula III is provided:

or a salt thereof, wherein V and R are as defined in Formula I. Incertain embodiments according to this paragraph, V is —W₁—W₂—; one of W₁and W₂ is absent or —CH₂—, and the other is —NH—, —O—, or —S—. Incertain embodiments, V is a single bond, —NII—, or —CH₂NH—. In certainembodiments, V is a single bond, —NH—, or —CH₂NH—; and R is methyl.

In an embodiment, a compound according to formula IV is provided:

or a salt thereof, wherein V and R are as defined in Formula I. Incertain embodiments according to this paragraph, V is —W₁—W₂—; one of W₁and W₂ is absent or —CH₂—, and the other is —NH—, —O—, or —S—. Incertain embodiments, V is a single bond, —NH—, or —CH₂NH—. In certainembodiments, V is a single bond, —NH—, or —CH₂NH—; and R is methyl.

In an embodiment, a compound according to formula V is provided:

or a salt thereof, wherein V and R are as defined in Formula I. Incertain embodiments according to this paragraph, V is —W₁—W₂—; one of W₁and W₂ is absent or —CH₂—, and the other is —NH—, —O—, or —S—. Incertain embodiments, V is a single bond, —NH—, or —CH₂NH—. In certainembodiments, V is a single bond, —NH—, or —CH₂NH—; and R is methyl.

In an embodiment, a compound according to formula VI is provided:

or a salt thereof, wherein V and R are as defined in Formula I. Incertain embodiments according to this paragraph, V is —W₁—W₂—; one of W₁and W₂ is absent or —CH₂—, and the other is —NH—, —O—, or —S—. Incertain embodiments, V is a single bond, —NH—, or —CH₂NH—. In certainembodiments, V is a single bond, —NH—, or —CH₂NH—; and R is methyl.

In an embodiment, a compound according to formula Vii is provided:

or a salt thereof, wherein V and R are as defined in Formula I. Incertain embodiments according to this paragraph, V is —W₁—W₂—; one of W₁and W₂ is absent or —CH₂—, and the other is —NH—, —O—, or —S—. Incertain embodiments, V is a single bond, —NH—, or —CH₂NH—. In certainembodiments, V is a single bond, —NH—, or —CH₂NH—; and R is methyl.

In an embodiment, a compound according to formula VIII is provided:

or a salt thereof, wherein V and R are as defined in Formula I. Incertain embodiments according to this paragraph, V is —W₁—W₂—; one of W₁and W₂ is absent or —CH₂—, and the other is —NH—, —O—, or —S—. Incertain embodiments, V is a single bond, —NH—, or —CH₂NH—. In certainembodiments, V is a single bond, —NH—, or —CH₂NH—; and R is methyl.

In an embodiment, a compound according to formula IX is provided:

or a salt thereof, wherein V and R are as defined in Formula I. Incertain embodiments according to this paragraph, V is —W₁—W₂—; one of W₁and W₂ is absent or —CH₂—, and the other is —NH—, —O—, or —S—. Incertain embodiments, V is a single bond, —NH—, or —CH₂NH—. In certainembodiments, V is a single bond, —NH—, or —CH₂NH—; and R is methyl.

In an embodiment, a compound according to any of formulas 1-10 isprovided:

or a salt thereof.

In an embodiment, a polypeptide is provided comprising an amino acidresidue corresponding to a compound of formula I, Ia, II, III, IV, V,VI, VII, VIII, IX, or 1-10. In an embodiment, a conjugate is providedcomprising a polypeptide comprising an amino acid residue correspondingto a compound of formula I, Ia, II, III, IV, V, VI, VII, VIII, IX, or1-10. linked to a payload and optionally comprising a linking moietybetween the polypeptide and the payload.

In an embodiment, an antibody is provided comprising an amino acidresidue corresponding to a compound of formula I, Ia, II, III, IV, V,VI, VII, VIII, IX, or 1-10. In an embodiment, a conjugate is providedcomprising an antibody comprising an amino acid residue corresponding toa compound of formula I, Ia, II, III, IV, V, VI, VII, VIII, IX, or 1-10.linked to a payload and optionally comprising a linking moiety betweenthe antibody and the payload.

In an embodiment, an orthogonal tRNA is provided aminoacylated with anamino acid residue corresponding to a compound of formula I, Ia, II,III, IV, V, VI, VII, VIII, IX, or 1-10. In a related embodiment, amethod of producing a polypeptide is provided, comprising contacting apolypeptide with an orthogonal tRNA aminoacylated with an amino acidresidue corresponding to a compound of formula I, Ia, II, III, IV, V,VI, VII, VIII, IX, or 1-10 under conditions suitable for incorporatingthe amino acid residue into the polypeptide. In an aspect, theorthogonal tRNA base pairs with a codon that is not normally associatedwith an amino acid. In another aspect, the contacting occurs in areaction mixture which comprises a tRNA synthetase capable ofaminoacylating the orthogonal tRNA with a compound of formula I, Ia, II,III, IV, V, VI, VII, VIII, IX, or 1-10.

In certain embodiments, a polypeptide comprising a modified amino acidresidue is provided according to any of the following formulas, whereAr, V and R are as defined in the context of formula I:

Those of skill in the art will recognize that proteins are generallycomprised of L-amino acid residues. However, with modified amino acids,the present methods and compositions provide the practitioner with theability to use L-, D- or racemic modified amino acids. In certainembodiments, the modified amino acids described herein includeD-versions of the natural amino acids and racemic versions of thenatural amino acids.

Tetrazine Ligation with Modified Amino Acids Comprising TetrazineFunctional Groups

Modification of amino acids to incorporate tetrazine functional groupsenables selective and efficient reaction of the modified amino acidswith compounds comprising strained alkenes. These reactions areselective in that the reactive groups—the tetrazines and the strainedalkenes—are not reactive with the functional groups of the naturallyoccurring amino acids or with other well-known reactive groups. Further,the reactions can be carried out in complex environments such as cellextracts, in vitro protein synthesis reaction mixtures and the like.

The reaction between tetrazine and a strained alkene is known as the“tetrazine ligation.” It is believed that the tetrazine and strainedalkene react in an inverse-demand Diels-Alder reaction followed by aretro-Diels-Alder reaction that links the tetrazine to the strainedalkene. The reaction is specific, with little to no cross-reactivitywith functional groups that occur on biomolecules. The reaction may becarried out under mild conditions, for example at room temperature andwithout a catalyst.

Useful strained alkenes include trans-cyclooctenes and norbornenesdescribed herein. Useful modified amino acids comprising tetrazinefunctional groups include compounds according to any of formulas I, Ia,II, III, IV, V, VI, VII, VIII, IX, or 1-10 described herein, andpolypeptides comprising residues corresponding to the compounds.

In some embodiments, modified amino acids comprising tetrazinefunctional groups are used in conjunction with modified amino acidscomprising other functional groups. In some embodiments, the modifiedamino acid comprising a tetrazine functional group is a modified aminoacid according to any of formulas I, Ia, II, III, IV, V, VI, VII, VIII,IX, or 1-10. This approach may be used, for example, to producepolypeptides or antibodies comprising modified amino acid residues withtwo or more different types of functional groups. In some embodiments,the polypeptides or antibodies comprise modified amino acids with threeor more different types of functional groups.

Useful additional functional groups for additional modified amino acidsinclude carbonyls, thiols, azides, alkynes, and other functional groupsdeemed suitable by those of skill in the art.

In advantageous embodiments, provided are polypeptides comprising one ormore of the modified amino acids comprising tetrazine moieties,described herein, along with one or more other modified amino acidscomprising an azide moiety. This combination of reactive amino acidsfacilitates two, independent reactions at specific sites on thepolypeptide. A molecule comprising a strained alkene can selectivelyreact with the one or more tetrazine moieties. Another moleculecomprising an alkyne group can react with the one or more azidemoieties. Advantageously, there can be little or no cross-reactionbetween the tetrazine ligations and the azide-alkyne condensations.

Incorporation of both tetrazine and azide functionality into a singlepolypeptide enables, for example, controlled conjugation of more thanone payload molecule to the polypeptide chain. For example, in someembodiments, a first payload comprises a strained alkene functionalgroup, enabling reaction with amino acid residues comprising tetrazine,while a second payload comprises an alkyne functional group, enablingreaction with amino acid residues comprising azide. Further payloads,comprising additional functional groups may also be used. The functionalgroups carried by such further payloads may react with any othersuitable functional group, such as a functional group on a furthermodified amino acid residue or a natural amino acid residue.

The reaction between an azido functional group and an alkyne, to form atriazole, is known as the Huisgen cycloaddition. It is believed that theazido and alkyne groups react in a 1,3-dipolar cycloaddition reaction toform a 1,2,3-triazolylene moiety which links the azido group to thealkyne. The reaction may be carried out at room temperature underaqueous conditions by the addition of Cu(II) (including but not limitedto, in the form of a catalytic amount of CuSO₄) in the presence of areducing agent for reducing Cu(II) to Cu(I), in situ, in catalyticamount. See, e.g., Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193(2003); Tornoe, C. W., et al., J. Org. Chem. 67:3057-3064 (2002);Rostovtsev, et al., Angew. Chem. Int. Ed. 41:2596-2599 (2002). Exemplaryreducing agents include, including but are not limited to, ascorbate,metallic copper, quinine, hydroquinone, vitamin K, glutathione,cysteine, Fe²⁺, Co²⁺, and an applied electric potential. In addition tothe Huisgen cycloaddition reaction, the azide functional group can bereacted selectively with a payload moiety comprising an aryl ester andan aryl phosphine moiety to generate an amide linkage. See Saxon andBertozzi, Science 287:2007-2010 (2000). The azide functional group canalso be reacted selectively with a payload moiety comprising a thioesterand an aryl phosphine moiety to generate an amide linkage.

Modification of amino acids to incorporate azide functional groupstherefore enables selective and efficient reaction of the modified aminoacids with molecules comprising alkynes, aryl esters and thioesters.Examples of modified amino acids comprising azide functional groupsinclude p-azido-phenylalanine (See Chin et al., J. Am. Chem. Soc.124:9026-9027 (2002)), as well as compounds according to any of formulasAI, A1-A30 or A40, as shown below. Such compounds may be prepared, forexample, according to methods provided in PCT/US2013/057677, which isincorporated by reference in its entirety.

In certain embodiments, the modified amino acid is a compound accordingto formula AI:

or a salt thereof, wherein: D is —Ar—W₃— or —W₁—Y₁—C(O)—Y₂—W₂—; Ar is

each of W₁, W₂, and W₃ is independently a single bond or lower alkylene;each X₁ is independently —NH—, —O—, or each Y₁ is independently a singlebond, —NH—, or —O—; each Y₂ is independently a single bond, —NH—, —O—,or an N-linked or C-linked pyrrolidinylene; and one of Z₁, Z₂, and Z₃ is—N— and the others of Z₁, Z₂, and Z₃ are independently —CH—. In certainembodiments, provided are polypeptides comprising modified tetrazineresidues, provided herein, along with residues according to one of thefollowing:

where D is as described above.

In some embodiments, the modified amino acid is a compound according toformula A1-A30 or A40:

In some embodiments, provided herein are polypeptides comprisingmodified tetrazine residues, provided herein, along with residuescorresponding to any of the modified amino acids p-azido-phenylalanine,A1-A30 or A40.

Polypeptides

Provided herein are polypeptides comprising one or more modified aminoacid residues at site-specific positions in an amino acid residuesequence of at least one polypeptide chain. In an embodiment, thecompositions are antibodies comprising one or more modified amino acidresidues at site-specific positions in the amino acid sequence of atleast one polypeptide chain.

In certain embodiments, a polypeptide comprises at least onetetrazine-comprising amino acid residue as described herein. In certainembodiments, a polypeptide comprises at least two tetrazine-comprisingamino acid residues as described herein. In certain embodiments, apolypeptide comprises at least three tetrazine-comprising amino acidresidues as described herein.

In certain embodiments, a polypeptide comprises at least onetetrazine-comprising amino acid residue as described herein and at leastone azide-comprising amino acid residue. In certain embodiments, apolypeptide comprises at least two tetrazine-comprising amino acidresidues as described herein and at least one azide-comprising aminoacid residue. In certain embodiments, a polypeptide comprises at leasttwo tetrazine-comprising amino acid residues as described herein and atleast two azide-comprising amino acid residues. In certain embodiments,a polypeptide comprises at least three tetrazine-comprising amino acidresidues as described herein and at least one azide-comprising aminoacid residue. In certain embodiments, a polypeptide comprises at leastthree tetrazine-comprising amino acid residues as described herein andat least two azide-comprising amino acid residues. In certainembodiments, a polypeptide comprises at least three tetrazine-comprisingamino acid residues as described herein and at least threeazide-comprising amino acid residues.

The polypeptide can share high sequence identity with any polypeptiderecognized by those of skill in the art, i.e. a parent polypeptide. Incertain embodiments, the amino acid residue sequence of the polypeptideis identical to the amino acid residue sequence of the parentpolypeptide, other than the modified amino acid residues atsite-specific positions. In further embodiments, the polypeptideprovided herein can have one or more insertions, deletions or mutationsrelative to the parent polypeptide in addition to the one or moremodified amino acid residues at the site-specific positions. In certainembodiments, the polypeptide provided herein can have a unique primarysequence, so long as it would be recognized as a polypeptide by those ofskill in the art. In certain aspects of this embodiment, the polypeptideis an antibody.

The compositions and methods described herein provide for theincorporation of at least one modified amino acid residue into apolypeptide. In some embodiments, at least two modified amino acidresidues are incorporated into a polypeptide. In some embodiments, atleast three modified amino acids residues are incorporated into apolypeptide. In some embodiments, at least four modified amino acidresidues are incorporated into a polypeptide. In some embodiments, atleast five modified amino acid residues are incorporated into apolypeptide. In some embodiments, at least six modified amino acidresidues are incorporated into a polypeptide. The modified amino acidresidues may be present at any location on the polypeptide, includingany terminal position or any internal position of the polypeptide.Preferably, the modified amino acid residue does not destroy theactivity and/or the tertiary structure of the polypeptide relative tothe homologous naturally-occurring polypeptide, unless such destructionof the activity and/or tertiary structure was one of the purposes ofincorporating the modified amino acid residue into the polypeptide.Further, the incorporation of the modified amino acid residue into thepolypeptide may modify to some extent the activity (e.g., manipulatingthe therapeutic effectiveness of the polypeptide, improving the safetyprofile of the polypeptide, adjusting the pharmacokinetics,pharmacologics and/or pharmacodynamics of the polypeptide (e.g.,increasing water solubility, bioavailability, increasing serumhalf-life, increasing therapeutic half-life, modulating immunogenicity,modulating biological activity, or extending the circulation time),providing additional functionality to the polypeptide, incorporating atag, label or detectable signal into the polypeptide, easing theisolation properties of the polypeptide, and any combination of theaforementioned modifications) and/or tertiary structure of thepolypeptide relative to the homologous naturally-occurring polypeptidewithout fully causing destruction of the activity and/or tertiarystructure. Such modifications of the activity and/or tertiary structureare often one of the goals of effecting such incorporations, althoughthe incorporation of the modified amino acid residue into thepolypeptide may also have little effect on the activity and/or tertiarystructure of the polypeptide relative to the homologousnaturally-occurring polypeptide. Correspondingly, polypeptidescomprising modified amino acid residues, compositions comprisingpolypeptides with modified amino acid residues, methods for making suchpolypeptides and polypeptide compositions, methods for purifying,isolating, and characterizing such polypeptides and polypeptidecompositions, and methods for using such polypeptides and polypeptidecompositions are considered within the scope of the present disclosure.Further, the polypeptides comprising modified amino acid residuesdescribed herein may also be ligated to other polypeptides (including,by way of example, a polypeptide comprising modified amino acid residuesor a naturally-occurring polypeptide).

The methods, compositions, strategies and techniques described hereinare not limited to a particular type, class or family of polypeptides orproteins. Indeed, virtually any polypeptide may include at least onemodified amino acid residue derived from the modified amino acidsdescribed herein. By way of example only, the polypeptide can behomologous to a therapeutic protein selected from the group consistingof: alpha-1 antitrypsin, angiostatin, antihemolytic factor, antibody,apolipoprotein, apoprotein, atrial natriuretic factor, atrialnatriuretic polypeptide, atrial peptide, C—X—C chemokine, T39765, NAP-2,ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG,calcitonin, c-kit ligand, cytokine, CC chemokine, monocytechemoattractant protein-1, monocyte chemoattractant protein-2, monocytechemoattractant protein-3, monocyte inflammatory protein-1 alpha,monocyte inflammatory protein-1β, RANTES, 1309, R83915, R91733, HCC1,T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen,colony stimulating factor (CSF), complement factor 5a, complementinhibitor, complement receptor 1, cytokine, epithelial neutrophilactivating peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF),epithelial neutrophil activating peptide, erythropoietin (EPO),exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X,fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helicalbundle protein, G-CSF, GLP-1, GM-CSF, glucocerebrosidase, gonadotropin,growth factor, growth factor receptor, GRF, hedgehog protein,hemoglobin, hepatocyte growth factor (hGF), hirudin, human growthhormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1,LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-1,IGF-II, interferon (IFN), IFN-β, IFN-γ, interleukin (IL), IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitoryfactor, luciferase, neurturin, neutrophil inhibitory factor (NIF),oncostatin M, osteogenic protein, oncogene product, paracitonin,parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin,protein A, protein G, PTH, pyrogenic exotoxin A, pyrogenic exotoxin B,pyrogenic exotoxin C, PYY, relaxin, renin, SCF, small biosyntheticprotein, soluble complement receptor I, soluble I-CAM 1, solubleinterleukin receptor, soluble TNF receptor, somatomedin, somatostatin,somatotropin, streptokinase, superantigens, staphylococcal enterotoxin,SEA, SEB, SECT, SEC2, SEC3, SED, SEE, steroid hormone receptor,superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1,tissue plasminogen activator, tumor growth factor (TGF), tumor necrosisfactor, tumor necrosis factor alpha, tumor necrosis factor beta, tumornecrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascularendothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53,tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor,testosterone receptor, aldosterone receptor, LDL receptor, andcorticosterone. In a related or further embodiment, the polypeptidecomprising a modified amino acid residue may also be homologous to anypolypeptide member of the growth hormone supergene family.

In certain embodiments, the modified amino acid residue can be at anyposition within the polypeptide, for example, at the N-terminus, at theC-terminus, or within the polypeptide. In advantageous embodiments, themodified amino acid residue is positioned at select locations in apolypeptide. These locations are identified as providing optimum sitesfor substitution with a modified amino acid residue. Each site iscapable of bearing a modified amino acid residue with optimum structure,function and/or methods for producing the polypeptide.

In certain embodiments, a site-specific position for substitutionprovides a polypeptide that is stable. Stability can be measured by anytechnique apparent to those of skill in the art.

In certain embodiments, a site-specific position for substitutionprovides a polypeptide that is has optimal functional properties. Forinstance, the polypeptide can show little or no loss of binding affinityfor its target compared to a polypeptide without the site-specificmodified amino acid residue. In certain embodiments, the polypeptide canshow enhanced binding compared to a polypeptide without thesite-specific modified amino acid residue. In certain aspects of thisembodiment, the polypeptide is an antibody and the target is an antigen.

In certain embodiments, a site-specific position for substitutionprovides a polypeptide that can be made advantageously. For instance, incertain embodiments, the polypeptide shows advantageous properties inits methods of synthesis, for example as discussed herein. In certainembodiments, the polypeptide can show little or no loss in yield inproduction compared to a polypeptide without the site-specific modifiedamino acid residue. In certain embodiments, the polypeptide can showenhanced yield in production compared to a polypeptide without thesite-specific modified amino acid residue. In certain embodiments, thepolypeptide can show little or no loss of tRNA suppression, for exampleas described herein, compared to a polypeptide without the site-specificmodified amino acid residue. In certain embodiments, the polypeptide canshow enhanced tRNA suppression, for example as described herein, inproduction compared to a polypeptide without the site-specific modifiedamino acid residue. In certain aspects of this embodiment, thepolypeptide is an antibody.

In certain embodiments, a site-specific position for substitutionprovides a polypeptide that has advantageous solubility. In certainembodiments, the polypeptide can show little or no loss in solubilitycompared to a polypeptide without the site-specific modified amino acidresidue. In certain embodiments, the polypeptide can show enhancedsolubility compared to a polypeptide without the site-specific modifiedamino acid residue. In certain aspects of this embodiment, thepolypeptide is an antibody.

In certain embodiments, a site-specific position for substitutionprovides a polypeptide that has advantageous expression. In certainembodiments, the polypeptide can show little or no loss in expressioncompared to a polypeptide without the site-specific modified amino acidresidue. In certain embodiments, the polypeptide can show enhancedexpression compared to a polypeptide without the site-specific modifiedamino acid residue. In certain aspects of this embodiment, thepolypeptide is an antibody.

In certain embodiments, a site-specific position for substitutionprovides a polypeptide that has advantageous folding. In certainembodiments, the polypeptide can show little or no loss in properfolding compared to a polypeptide without the site-specific modifiedamino acid residue. In certain embodiments, the polypeptide can showenhanced folding compared to a polypeptide without the site-specificmodified amino acid residue. In certain aspects of this embodiment, thepolypeptide is an antibody.

In certain embodiments, a site-specific position for substitutionprovides a polypeptide that is capable of advantageous conjugation. Asdescribed herein, several modified amino acid residues have side chainsor functional groups that facilitate conjugation of the polypeptide toone or more agents, either directly or via a linker. In certainembodiments, the polypeptide can show enhanced conjugation efficiencycompared to a polypeptide without the same or other modified amino acidresidues at other positions. In certain embodiments, the polypeptide canshow enhanced conjugation yield compared to a polypeptide without thesame or other modified amino acid residues at other positions. Incertain embodiments, the polypeptide can show enhanced conjugationspecificity compared to a polypeptide without the same or other modifiedamino acid residues at other positions. In certain aspects of thisembodiment, the polypeptide is an antibody.

In certain embodiments, further provided herein are conservativelymodified variants of the polypeptides described herein. Conservativelymodified variants of a polypeptide include one or more insertions,deletions or substitutions that do not disrupt the structure and/orfunction of the polypeptide when evaluated by one of skill in the art.In certain embodiments, conservatively modified variants include 20 orfewer amino acid residue insertions, deletions or substitutions. Incertain embodiments, conservatively modified variants include 15 orfewer amino acid residue insertions, deletions or substitutions. Incertain embodiments, conservatively modified variants include 10 orfewer amino acid residue insertions, deletions or substitutions. Incertain embodiments, conservatively modified variants include 9 or feweramino acid residue insertions, deletions or substitutions. In certainembodiments, conservatively modified variants include 8 or fewer aminoacid residue insertions, deletions or substitutions. In certainembodiments, conservatively modified variants include 7 or fewer aminoacid residue insertions, deletions or substitutions. In certainembodiments, conservatively modified variants include 6 or fewer aminoacid residue insertions, deletions or substitutions. In certainembodiments, conservatively modified variants include 5 or fewer aminoacid residue insertions, deletions or substitutions. In certainembodiments, conservatively modified variants include 4 or fewer aminoacid residue insertions, deletions or substitutions. In certainembodiments, conservatively modified variants include 3 or fewer aminoacid residue insertions, deletions or substitutions. In certainembodiments, conservatively modified variants include 2 or fewer aminoacid residue insertions, deletions or substitutions. In certainembodiments, conservatively modified variants include 1 amino acidresidue insertion, deletion or substitution. In particular embodimentsthe substitutions are conservative, substituting an amino acid residuewithin the same class, as described herein. In particular embodiments,the polypeptide is an antibody.

In certain embodiments, the polypeptides can be modified to modulatestructure, stability and/or activity. In such embodiments, themodifications can be conservative or non-conservative. The modificationsneed only be suitable to the practitioner carrying out the methods andusing the compositions described herein. In certain embodiments wherethe polypeptide is an antibody, the modifications decrease but do noteliminate antigen binding affinity. In certain embodiments where thepolypeptide is an antibody, the modifications increase antigen bindingaffinity. In certain embodiments, the modifications enhance structure orstability of the polypeptide. In certain embodiments, the modificationsreduce but do not eliminate structure or stability of the polypeptide.In certain embodiments, modified variants include 20 or fewer amino acidresidue insertions, deletions or substitutions. In certain embodiments,modified variants include 15 or fewer amino acid residue insertions,deletions or substitutions. In certain embodiments, modified variantsinclude 10 or fewer amino acid residue insertions, deletions orsubstitutions. In certain embodiments, modified variants include 9 orfewer amino acid residue insertions, deletions or substitutions. Incertain embodiments, modified variants include 8 or fewer amino acidresidue insertions, deletions or substitutions. In certain embodiments,modified variants include 7 or fewer amino acid residue insertions,deletions or substitutions. In certain embodiments, modified variantsinclude 6 or fewer amino acid residue insertions, deletions orsubstitutions. In certain embodiments, modified variants include 5 orfewer amino acid residue insertions, deletions or substitutions. Incertain embodiments, modified variants include 4 or fewer amino acidresidue insertions, deletions or substitutions. In certain embodiments,modified variants include 3 or fewer amino acid residue insertions,deletions or substitutions. In certain embodiments, modified variantsinclude 2 or fewer amino acid residue insertions, deletions orsubstitutions. In certain embodiments, modified variants include 1 aminoacid residue insertion, deletion or substitution.

Also within the scope are post-translationally modified variants. Any ofthe polypeptides provided herein can be post-translationally modified inany manner recognized by those of skill in the art. Typicalpost-translational modifications for polypeptides include interchaindisulfide bonding, intrachain disulfide bonding, N-linked glycosylationand proteolysis. Also provided herein are other post-translationallymodified polypeptides having modifications such as phosphorylation,O-linked glycosylation, methylation, acetylation, lipidation, GPIanchoring, myristoylation and prenylation. The post-translationalmodification can occur during production, in vivo, in vitro orotherwise. In certain embodiments, the post-translational modificationcan be an intentional modification by a practitioner, for instance,using the methods provided herein. In particular embodiments, thepolypeptide is an antibody.

Further included are polypeptides fused to further peptides orpolypeptides. Exemplary fusions include, but are not limited to, e.g., amethionyl polypeptide in which a methionine is linked to the N-terminusof the polypeptide resulting from the recombinant expression, fusionsfor the purpose of purification (including but not limited to, topoly-histidine or affinity epitopes), fusions for the purpose of linkingto other biologically active molecules, fusions with serum albuminbinding peptides, and fusions with serum proteins such as serum albumin.The polypeptides may comprise protease cleavage sequences, reactivegroups, polypeptide-binding domains (including but not limited to, FLAGor poly-His) or other affinity based sequences (including but notlimited to e.g., GST). The polypeptides may also comprise linkedmolecules (including but not limited to, biotin) that improve detection(including but not limited to, GFP), purification or other features ofthe polypeptide. In certain embodiments, the polypeptides comprise aC-terminal affinity sequence that facilitates purification of fulllength polypeptides. In certain embodiments, such C-terminal affinitysequence is a poly-His sequence, e.g., a 6-His sequence. In particularembodiments, the polypeptide is an antibody.

Also provided herein are polypeptides that are conjugated to one or moreconjugation moieties. The conjugation moiety can be any conjugationmoiety deemed useful to one of skill in the art. For instance, theconjugation moiety can be a polymer, such as polyethylene glycol, thatcan improve the stability of the polypeptide in vitro or in vivo. Theconjugation moiety can have therapeutic activity, thereby yielding apolypeptide-drug conjugate. The conjugation moiety can be a payload thatis harmful to target cells. The conjugation moiety can be a label usefulfor detection or diagnosis. In certain embodiments, the conjugationmoiety is linked to the polypeptide via a direct covalent bond. Incertain embodiments, the conjugation moiety is linked to the polypeptidevia a linker. In advantageous embodiments, the conjugation moiety or thelinker is attached via one of the modified amino acid residues of thepolypeptide. Exemplary conjugation moieties and linkers are describedherein. In certain embodiments, the conjugation moiety is linked to thepolypeptide via a non-covalent interaction. In particular embodiments,the polypeptide is an antibody.

The parent polypeptide can be any polypeptide known to those of skill inthe art, or later discovered, without limitation. In particularembodiments, the polypeptide is an antibody. The parent polypeptide maybe substantially encoded by a polypeptide gene or polypeptide genes fromany organism, including but not limited to humans, mice, rats, rabbits,chickens, camels, llamas, dromedaries, monkeys, particularly mammals andparticularly human and particularly mice and rats. In one embodiment,the parent polypeptide may be fully human, obtained for example from apatient or subject, by using transgenic mice or other animals (seeBruggemann & Taussig, 1997, Curr. Opin. Biotechnol. 8:455-458 forantibody examples) or human polypeptide libraries coupled with selectionmethods (see Griffiths & Duncan, 1998, Curr. Opin. Biotechnol. 9:102-108for antibody examples). The parent polypeptide may be from any source,including artificial or naturally occurring. For example, a parentpolypeptide can be an engineered polypeptide, including but not limitedto chimeric polypeptides and humanized polypeptides (see Clark, 2000,Immunol. Today 21:397-402 for antibody examples) or derived from acombinatorial library. In addition, the parent polypeptide may be anengineered variant of a polypeptide that is substantially encoded by oneor more natural polypeptide genes. In some embodiments, the parentpolypeptide is a polypeptide that has been identified by affinitymaturation.

Parent polypeptides can be any polypeptide known in the art or anypolypeptide developed by those of skill in the art without limitation.Antibody examples include, but are not limited to anti-TNF antibody(U.S. Pat. No. 6,258,562), anti-IL-12 and/or anti-IL-12p40 antibody(U.S. Pat. No. 6,914,128); anti-IL-18 antibody (U.S. Patent PublicationNo. 2005/0147610), anti-05, anti-CBL, anti-CD147, anti-gp120,anti-VLA-4, anti-CD11a, anti-CD18, anti-VEGF, anti-CD40L, anti CD-40(e.g., see PCT Publication No. WO 2007/124299) anti-Id, anti-ICAM-1,anti-CXCL13, anti-CD2, anti-EGFR, anti-TGF-β2, anti-HGF, anti-cMet, antiDLL-4, anti-NPR1, anti-PLGF, anti-ErbB3, anti-E-selectin, anti-Fact VII,anti-Her2/neu, anti-F gp, anti-CD11/18, anti-CD14, anti-ICAM-3,anti-RON, anti-SOST, anti CD-19, anti-CD80 (e.g., see PCT PublicationNo. WO 2003/039486, anti-CD4, anti-CD3, anti-CD23, anti-β2-integrin,anti-α4β7, anti-CD52, anti-HLA DR, anti-CD22 (e.g., see U.S. Pat. No.5,789,554), anti-CD20, anti-MIF, anti-CD64 (FcR), anti-TCR α and/or β,anti-CD2, anti-Hep B, anti-CA 125, anti-EpCAM, anti-gp120, anti-CMV,anti-gpIIbIIIa, anti-IgE, anti-CD25, anti-CD33, anti-HLA, anti-IGF1,2,anti-IGFR, anti-VNRintegrin, anti-IL-1α, anti-IL-1β, anti-IL-1 receptor,anti-IL-2 receptor, anti-IL-4, anti-IL-4 receptor, anti-IL5, anti-IL-5receptor, anti-IL-6, anti-IL-8, anti-IL-9, anti-IL-13, anti-IL-13receptor, anti-IL-17, anti-IL-6R, anti-RANKL, anti-NGF, anti-DKK,anti-αVβ3, anti-IL-17A, anti-IL23p19 and anti-IL-23 (see Presta, L. G.(2005) J. Allergy Clin. Immunol. 116: 731-6).

Parent polypeptides may also be selected from various therapeuticpolypeptides approved for use, in clinical trials, or in development forclinical use. Antibody examples of such therapeutic polypeptidesinclude, but are not limited to, rituximab (Rituxan®,IDEC/Genentech/Roche) (see, for example, U.S. Pat. No. 5,736,137), achimeric anti-CD20 antibody approved to treat Non-Hodgkin's lymphoma;HuMax-CD20, an anti-CD20 currently being developed by Genmab, ananti-CD20 antibody described in U.S. Pat. No. 5,500,362, AME-133(Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM(Intracel), and PRO70769 (PCT Application No. PCT/US2003/040426),trastuzumab (Herceptin®, Genentech) (see, for example, U.S. Pat. No.5,677,171), a humanized anti-Her2/neu antibody approved to treat breastcancer; pertuzumab (rhuMab-2C4, Omnitarg®), currently being developed byGenentech; an anti-Her2 antibody (U.S. Pat. No. 4,753,894; cetuximab(Erbitux®, Imclone) (U.S. Pat. No. 4,943,533; PCT Publication No. WO96/40210), a chimeric anti-EGFR antibody in clinical trials for avariety of cancers; ABX-EGF (U.S. Pat. No. 6,235,883), currently beingdeveloped by Abgenix-Immunex-Amgen; HuMax-EGFr (U.S. Pat. No.7,247,301), currently being developed by Genmab; 425, EMD55900,EMD62000, and EMD72000 (Merck KGaA) (U.S. Pat. No. 5,558,864; Murthy, etal. (1987) Arch. Biochem. Biophys. 252(2): 549-60; Rodeck, et al. (1987)J. Cell. Biochem. 35(4): 315-20; Kettleborough, et al. (1991) ProteinEng. 4(7): 773-83); ICR62 (Institute of Cancer Research) (PCTPublication No. WO 95/20045; Modjtahedi, et al. (1993) J. Cell. Biophys.22(1-3): 129-46; Modjtahedi, et al. (1993) Br. J. Cancer 67(2): 247-53;Modjtahedi, et al. (1996) Br. J. Cancer 73(2): 228-35; Modjtahedi, etal. (2003) Int. J. Cancer 105(2): 273-80); TheraCIM hR3 (YM Biosciences,Canada and Centro de Immunologia Molecular, Cuba (U.S. Pat. No.5,891,996; U.S. Pat. No. 6,506,883; Mateo, et al. (1997) Immunotechnol.3(1): 71-81); mAb-806 (Ludwig Institute for Cancer Research, MemorialSloan-Kettering) (Jungbluth, et al. (2003) Proc. Natl. Acad. Sci. USA.100(2): 639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX, National CancerInstitute) (PCT Publication No. WO 01/62931A2); and SC₁₀₀ (Scancell)(PCT Publication No. WO 01/88138); alemtuzumab (Campath®, Millenium), ahumanized mAb currently approved for treatment of B-cell chroniclymphocytic leukemia; muromonab-CD3 (Orthoclone OKT3®), an anti-CD3antibody developed by Ortho Biotech/Johnson & Johnson, ibritumomabtiuxetan (Zevalin®), an anti-CD20 antibody developed by IDEC/ScheringAG, gemtuzumab ozogamicin (Mylotarg®), an anti-CD33 (p67 protein)antibody developed by Celltech/Wyeth, alefacept (Amevive®), ananti-LFA-3 Fc fusion developed by Biogen), abciximab (ReoPro®),developed by Centocor/Lilly, basiliximab (Simulect®), developed byNovartis, palivizumab (Synagis®), developed by Medimmune, infliximab(Remicade®), an anti-TNFα antibody developed by Centocor, adalimumab(Humira®), an anti-TNFα antibody developed by Abbott, Humicade®, ananti-TNFα antibody developed by Celltech, golimumab (CNTO-148), a fullyhuman TNF antibody developed by Centocor, etanercept (Enbrel®), an p75TNF receptor Fc fusion developed by Immunex/Amgen, Ienercept, an p55TNFreceptor Fc fusion previously developed by Roche, ABX-CBL, an anti-CD147antibody being developed by Abgenix, ABX-IL8, an anti-IL8 antibody beingdeveloped by Abgenix, ABX-MA1, an anti-MUC18 antibody being developed byAbgenix, Pemtumomab (R1549, 90Y-muHMFG1), an anti-MUC1 in development byAntisoma, Therex (R1550), an anti-MUC1 antibody being developed byAntisoma, AngioMab (AS1405), being developed by Antisoma, HuBC-1, beingdeveloped by Antisoma, Thioplatin (AS1407) being developed by Antisoma,Antegren® (natalizumab), an anti-α-4-β-1 (VLA-4) and α-4-β-7 antibodybeing developed by Biogen, VLA-1 mAb, an anti-VLA-1 integrin antibodybeing developed by Biogen, LTBR mAb, an anti-lymphotoxin beta receptor(LTBR) antibody being developed by Biogen, CAT-152, an anti-TGF-βantibody being developed by Cambridge Antibody Technology, ABT 874(J695), an anti-IL-12 p40 antibody being developed by Abbott, CAT-192,an anti-TGFβ1 antibody being developed by Cambridge Antibody Technologyand Genzyme, CAT-213, an anti-Eotaxinl antibody being developed byCambridge Antibody Technology, LymphoStat-B® an anti-Blys antibody beingdeveloped by Cambridge Antibody Technology and Human Genome SciencesInc., TRAIL-R1 mAb, an anti-TRAIL-R1 antibody being developed byCambridge Antibody Technology and Human Genome Sciences, Inc., Avastin®bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody being developed byGenentech, an anti-HER receptor family antibody being developed byGenentech, Anti-Tissue Factor (ATF), an anti-Tissue Factor antibodybeing developed by Genentech, Xolair® (Omalizumab), an anti-IgE antibodybeing developed by Genentech, Raptiva® (Efalizumab), an anti-CD11aantibody being developed by Genentech and Xoma, MLN-02 Antibody(formerly LDP-02), being developed by Genentech and MilleniumPharmaceuticals, HuMax CD4, an anti-CD4 antibody being developed byGenmab, HuMax-1L15, an anti-1L15 antibody being developed by Genmab andAmgen, HuMax-Inflam, being developed by Genmab and Medarex,HuMax-Cancer, an anti-Heparanase I antibody being developed by Genmaband Medarex and Oxford GcoSciences, HuMax-Lymphoma, being developed byGenmab and Amgen, HuMax-TAC, being developed by Genmab, IDEC-131, andanti-CD40L antibody being developed by IDEC Pharmaceuticals, IDEC-151(Clenoliximab), an anti-CD4 antibody being developed by IDECPharmaceuticals, IDEC-114, an anti-CD80 antibody being developed by IDECPharmaceuticals, IDEC-152, an anti-CD 23 being developed by IDECPharmaceuticals, anti-macrophage migration factor (MIF) antibodies beingdeveloped by IDEC Pharmaceuticals, BEC2, an anti-idiotypic antibodybeing developed by Imclone, IMC-1C11, an anti-KDR antibody beingdeveloped by Imclone, DC₁₀₁, an anti-flk-1 antibody being developed byImclone, anti-VE cadherin antibodies being developed by Imclone,CEA-Cide® (Iabetuzumab), an anti-carcinoembryonic antigen (CEA) antibodybeing developed by Immunomedics, LymphoCide® (Epratuzumab), an anti-CD22antibody being developed by Immunomedics, AFP-Cide, being developed byImmunomedics, MyelomaCide, being developed by Immunomedics, LkoCide,being developed by Immunomedics, ProstaCide, being developed byImmunomedics, MDX-010, an anti-CTLA4 antibody being developed byMedarex, MDX-060, an anti-CD30 antibody being developed by Medarex,MDX-070 being developed by Medarex, MDX-018 being developed by Medarex,Osidem® (IDM-1), and anti-Her2 antibody being developed by Medarex andImmuno-Designed Molecules, HuMax®-CD4, an anti-CD4 antibody beingdeveloped by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibody beingdeveloped by Medarex and Genmab, CNTO 148, an anti-TNFα antibody beingdeveloped by Medarex and Centocor/J&J, CNTO 1275, an anti-cytokineantibody being developed by Centocor/J&J, MOR101 and MOR102,anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies beingdeveloped by MorphoSys, MOR201, an anti-fibroblast growth factorreceptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion®(visilizumab), an anti-CD3 antibody being developed by Protein DesignLabs, HuZAF®, an anti-gamma interferon antibody being developed byProtein Design Labs, Anti-α5β1 Integrin, being developed by ProteinDesign Labs, anti-IL-12, being developed by Protein Design Labs, ING-1,an anti-Ep-CAM antibody being developed by Xoma, Xolair® (Omalizumab) ahumanized anti-IgE antibody developed by Genentech and Novartis, andMLN01, an anti-Beta2 integrin antibody being developed by Xoma. In someembodiments, the therapeutics include KRN330 (Kirin); huA33 antibody(A33, Ludwig Institute for Cancer Research); CNTO 95 (alpha V integrins,Centocor); MEDI-522 (αVβ3integrin, Medimmune); volociximab (αVβ1integrin, Biogen/PDL); Human mAb 216 (B cell glycosolated epitope, NCI);BITE MT103 (bispecific CD19×CD3, Medimmune); 4G7×H22 (BispecificBcellxFcγR1, Medarex/Merck KGa); rM28 (Bispecific CD28×MAPG, EP PatentNo. EP1444268); MDX447 (EMD 82633) (Bispecific CD64×EGFR, Medarex);Catumaxomab (removab) (Bispecific EpCAM× anti-CD3, Trion/Fres);Ertumaxomab (bispecific HER2/CD3, Fresenius Biotech); oregovomab(OvaRex) (CA-125, ViRexx); Rencarex® (WX G250) (carbonic anhydrase IX,Wilex); CNTO 888 (CCL2, Centocor); TRC₁₀₅ (CD105 (endoglin), Tracon);BMS-663513 (CD137 agonist, Brystol Myers Squibb); MDX-1342 (CD19,Medarex); Siplizumab (MEDI-507) (CD2, Medimmune); Ofatumumab(Humax-CD20) (CD20, Genmab); Rituximab (Rituxan) (CD20, Genentech);veltuzumab (hA20) (CD20, Immunomedics); Epratuzumab (CD22, Amgen);lumiliximab (IDEC 152) (CD23, Biogen); muromonab-CD3 (CD3, Ortho);HuM291 (CD3 fc receptor, PDL Biopharma); HeFi-1, CD30, NCI); MDX-060(CD30, Medarex); MDX-1401 (CD30, Medarex); SGN-30 (CD30, SeattleGenentics); SGN-33 (Lintuzumab) (CD33, Seattle Genentics); Zanolimumab(HuMax-CD4) (CD4, Genmab); HCD122 (CD40, Novartis); SGN-40 (CD40,Seattle Genentics); Campathlh (Alemtuzumab) (CD52, Genzyme); MDX-1411(CD70, Medarex); hLL1 (EPB-1) (CD74.38, Immunomedics); Galiximab(IDEC-144) (CD80, Biogen); MT293 (TRC093/D93) (cleaved collagen,Tracon); HuLuc63 (CS1, PDL Pharma); ipilimumab (MDX-010) (CTLA4, BrystolMyers Squibb); Tremelimumab (Ticilimumab, CP-675,2) (CTLA4, Pfizer);HGS-ETR1 (Mapatumumab) (DR4TRAIL-R1 agonist, Human Genome Science/GlaxoSmith Kline); AMG-655 (DR5, Amgen); Apomab (DR5, Genentech); CS-1008(DR5, Daiichi Sankyo); HGS-ETR2 (lexatumumab) (DR5TRAIL-R2 agonist,HGS); Cetuximab (Erbitux) (EGFR, Imclone); IMC-11F8, (EGFR, Imclone);Nimotuzumab (EGFR, YM Bio); Panitumumab (Vectabix) (EGFR, Amgen);Zalutumumab (HuMaxEGFr) (EGFR, Genmab); CDX-110 (EGFRvIII, AVANTImmunotherapeutics); adecatumumab (MT201) (Epcam, Merck); edrecolomab(Panorex, 17-1A) (Epcam, Glaxo/Centocor); MORAb-003 (folate receptor a,Morphotech); KW-2871 (ganglioside GD3, Kyowa); MORAb-009 (GP-9,Morphotech); CDX-1307 (MDX-1307) (hCGb, Celldex); Trastuzumab(Herceptin) (HER2, Celldex); Pertuzumab (rhuMAb 2C4) (HER2 (DI),Genentech); apolizumab (HLA-DR β chain, PDL Pharma); AMG-479 (IGF-1R,Amgen); anti-IGF-1R R1507 (IGF1-R, Roche); CP 751871 (IGF1-R, Pfizer);IMC-A12 (IGF1-R, Imclone); BIIB022 (IGF-1R, Biogen); Mik-β-1 (IL-2Rb(CD122), Hoffman LaRoche); CNTO 328 (IL6, Centocor); Anti-KIR (1-7F9)(Killer cell Ig-like Receptor (KIR), Novo); Hu3S193 (Lewis (y), Wyeth,Ludwig Institute of Cancer Research); hCBE-11 (LTβR, Biogen); HuHMFG1(MUC1, Antisoma/NC1); RAV12 (N-linked carbohydrate epitope, Raven); CAL(parathyroid hormone-related protein (PTH-rP), University ofCalifornia); CT-011 (PD1, CureTech); MDX-1106 (ono-4538) (PD1,Medarex/Ono); MAb CT-011 (PD1, Curetech); IMC-3G3 (PDGFRa, Imclone);bavituximab (phosphatidylserine, Peregrine); huJ591 (PSMA, CornellResearch Foundation); muJ591 (PSMA, Cornell Research Foundation); GC₁₀₀₈(TGFb (pan) inhibitor (IgG4), Genzyme); Infliximab (Remicade) (TNFa,Centocor); A27.15 (transferrin receptor, Salk Institute, INSERN WO2005/111082); E2.3 (transferrin receptor, Salk Institute); Bevacizumab(Avastin) (VEGF, Genentech); HuMV833 (VEGF, Tsukuba Research Lab, PCTPublication No. WO/2000/034337, University of Texas); IMC-18F1 (VEGFR1,Imclone); IMC-1121 (VEGFR2, Imclone).

In certain embodiments, the polypeptides provided herein comprise onemodified amino acid residue at a site-specific position. In certainembodiments, the polypeptides provided herein comprise two modifiedamino acid residues at site-specific positions. In certain embodiments,the polypeptides provided herein comprise three modified amino acidresidues at site-specific positions. In certain embodiments, thepolypeptides provided herein comprise more than three modified aminoacid residues at site-specific positions.

Antibodies

In certain embodiments, provided herein are antibodies comprising one ormore polypeptides that comprise one or more modified amino acid residuesdescribed herein. In certain embodiments, the antibody is aheterotetramer comprising two identical light (L) chains and twoidentical heavy (H) chains. In some embodiments, incorporation ofdifferent light chains and/or heavy chains may be used to produceantibodies with more than one specificity (e.g., bispecificity). Thus,in certain embodiments, the antibody comprises two different lightchains and a single heavy chain. In some embodiments, the antibodycomprises two different heavy chains and a single light chain. In someembodiments the antibody comprises two different light chains and twodifferent heavy chains.

Each light chain can be linked to a heavy chain by a disulfide bond.Each heavy chain can be linked to the other heavy chain by one or moredisulfide bonds. Each heavy chain and each light chain can also have oneor more intrachain disulfide bonds. As is known to those of skill in theart, each heavy chain typically comprises a variable domain (V_(H))followed by a number of constant domains. Each light chain typicallycomprises a variable domain (V_(L)) and a constant domain. As is knownto those of skill in the art, antibodies typically have selectiveaffinity for their target molecules, i.e. antigens.

The antibodies provided herein can have any polypeptide form known tothose of skill in the art. They can be full-length or fragments.Exemplary full length antibodies include IgA, IgA1, IgA2, IgD, IgE, IgG,IgG1, IgG2, IgG3, IgG4, and IgM. Exemplary fragments include Fv, Fab,Fc, sFv, and other fragments known in the art and discussed herein.

The one or more modified amino acid residues can be located at selectedsite-specific positions in at least one polypeptide chain of anantibody. The polypeptide chain can be any polypeptide chain of theantibody without limitation, including either light chain or eitherheavy chain. The site-specific position can be in any domain of theantibody, including any variable domain and any constant domain.

Examples of Site-Specific Positions for Substitution: Heavy Chains

In this disclosure, the Kabat or Chothia numbering system is used torefer to amino acid residues in positions 117 or less of a heavy chainor heavy chain variable region, and positions 107 or less of a lightchain or light chain variable region. As is customary in the art, the EUnumbering system is used to refer to amino acid residues in positions118 or greater of a heavy chain and positions 108 or greater of a lightchain.

The site-specific positions for substituting can be described with anypolypeptide nomenclature system known to those of skill in the art,including the Kabat or Chothia numbering systems, the EU numberingsystem, or by reference to an amino acid residue or nucleotide sequence(e.g., by reference to SEQ ID NO:1 or SEQ ID NO:2, representing anexemplary heavy chain and an exemplary light chain, respectively). Table1 provides the correspondence between Kabat, Chothia, and EU residuenumbers, and the residue numbers in SEQ ID NO:1 and SEQ ID NO: 2. Theterm “minus 1”, when referring to Kabat or Chothia numbers, refers to anamino acid residue that directly precedes the first (i.e., H001 or L001)residue of the heavy chain or the light chain. Residue “minus 1” mayoccur, for example, in a sequence that is part of a leader sequence thatis N-terminal to the heavy chain or light chain. Such leader sequencesmay be naturally occurring or engineered.

In an embodiment wherein the polypeptide is an antibody, thesite-specific positions are selected from heavy chain residues H005,H023, H042, H065, H074, and H084 according to the Kabat or Chothianumbering schemes; and residues H118, H119, H132, H134, H135, H136,H137, H138, H139, H155, H160, H162, H165, H172, H174, H176, H177, H191,H194, H219, H238, H239, H241, H243, H246, H262, H264, H265, H267, H268,H269, H270, H271, H272, H274, H275, H278, H280, H281, H282, H283, H286,H289, H292, H293, H294, H295, H296, H297, H298, H299, H300, H301, H303,H305, H317, H320, H324, H326, H327, H329, H330, H332, H333, H334, H335,H337, H339, H340, H344, H355, H356, H358, H359, H360, H375, H383, H384,H386, H389, H392, H398, H420, H421, H436, and H438 according to the EUnumbering scheme (see Table 2, “SSHC-1” and the corresponding positionsof the representative heavy chain polypeptide according to SEQ 1D NO:1).Specifically provided herein are antibodies comprising one or moremodified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia heavy chain residues H005, H023, H074, and H084; and EUheavy chain residues H118, H119, H132, H134, H135, H136, H137, H139,H160, H162, H165, H172, H191, H194, H239, H241, H246, H267, H268, H269,H270, H271, H272, H274, H275, H280, H281, H282, H283, H286, H289, H292,H293, H294, H295, H296, H297, H298, H299, H300, H301, H303, H305, H317,H320, H324, H326, H327, H329, H330, H332, H333, H334, H335, H337, H339,H340, H344, H355, H359, H375, H386, H389, H392, H398, H420, H421, andH438 (see Table 2, “SSHC-2” and the corresponding positions of therepresentative heavy chain polypeptide according to SEQ ID NO:1).Specifically provided herein are antibodies comprising one or moremodified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia heavy chain residues H005 and H084; and EU heavy chainresidues H118, H132, H136, H239, H293, H334, H355, H359, and H389 (seeTable 2, “SSHC-3” and the corresponding positions of the representativeheavy chain polypeptide according to SEQ ID NO:1). Specifically providedherein are antibodies comprising one or more modified amino acidresidues at one or more of these positions; antibodies comprising one ormore modified amino acid residues at two or more of these positions;antibodies comprising one or more modified amino acid residues at threeor more of these positions; antibodies comprising two or more modifiedamino acid residues at one or more of these positions; antibodiescomprising two or more modified amino acid residues at two or more ofthese positions; antibodies comprising two or more modified amino acidresidues at three or more of these positions; antibodies comprisingthree or more modified amino acid residues at one or more of thesepositions; antibodies comprising three or more modified amino acidresidues at two or more of these positions; and antibodies comprisingthree or more modified amino acid residues at three or more of thesepositions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia heavy chain residues H023 and H074; and EU heavy chainresidues H119, H134, H135, H137, H139, H160, H162, H165, H172, H191,H194, H241, H246, H267, H268, H269, H270, H271, H272, H274, H275, H280,H281, H282, H283, H286, H289, H292, H294, H295, H296, H297, H298, H299,H300, H301, H303, H305, H317, H320, H324, H326, H327, H329, H330, H332,H333, H335, H337, H339, H340, H344, H355, H375, H386, H392, H398, H420,H421, and H438 (see Table 2, “SSHC-4” and the corresponding positions ofthe representative heavy chain polypeptide according to SEQ ID NO:1).Specifically provided herein are antibodies comprising one or moremodified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues H042 and H065; and EU residues H138, H155,H174, H176, H177, H219, H238, H243, H262, H264, H265, H278, H356, H358,H360, H383, H384 and H436 (see Table 2, “SSHC-5” and the correspondingpositions of the representative heavy chain polypeptide according to SEQID NO:1). Specifically provided herein are antibodies comprising one ormore modified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromEU residues corresponding to H292, H293, H294, H295, H296, H297, H298,H299, H300, H301, H303, and H305 (see Table 2, “SSHC-6” and thecorresponding positions of the representative heavy chain polypeptideaccording to SEQ ID NO:1). Specifically provided herein are antibodiescomprising one or more modified amino acid residues at one or more ofthese positions; antibodies comprising one or more modified amino acidresidues at two or more of these positions; antibodies comprising one ormore modified amino acid residues at three or more of these positions;antibodies comprising two or more modified amino acid residues at one ormore of these positions; antibodies comprising two or more modifiedamino acid residues at two or more of these positions; antibodiescomprising two or more modified amino acid residues at three or more ofthese positions; antibodies comprising three or more modified amino acidresidues at one or more of these positions; antibodies comprising threeor more modified amino acid residues at two or more of these positions;and antibodies comprising three or more modified amino acid residues atthree or more of these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H019, H025, H040, H052, H071,and H117; and EU residues corresponding to H119, H124, H139, H183, H193,H224, H225, and H407 (see Table 2, “SSHC-7” and the correspondingpositions of the representative heavy chain polypeptide according to SEQID NO:1). Specifically provided herein are antibodies comprising one ormore modified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H019, H025, H040, H052, andH070; and EU residues corresponding to H119, H121, H136, H180, H190,H222, H241, and H404 (see Table 2, “SSHC-8” and the correspondingpositions of the representative heavy chain polypeptide according to SEQID NO:1). Specifically provided herein are antibodies comprising one ormore modified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H025 and H040; and EUresidues corresponding to H119, H121, H136, H180, H190, H222, H241, andH404 (see Table 2, “SSHC-9” and the corresponding positions of therepresentative heavy chain polypeptide according to SEQ ID NO:1).Specifically provided herein are antibodies comprising one or moremodified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromEU residues corresponding to H121, H136, H180, H241, and H404 (see Table2, “SSHC-10” and the corresponding positions of the representative heavychain polypeptide according to SEQ ID NO:1). Specifically providedherein are antibodies comprising one or more modified amino acidresidues at one or more of these positions; antibodies comprising one ormore modified amino acid residues at two or more of these positions;antibodies comprising one or more modified amino acid residues at threeor more of these positions; antibodies comprising two or more modifiedamino acid residues at one or more of these positions; antibodiescomprising two or more modified amino acid residues at two or more ofthese positions; antibodies comprising two or more modified amino acidresidues at three or more of these positions; antibodies comprisingthree or more modified amino acid residues at one or more of thesepositions; antibodies comprising three or more modified amino acidresidues at two or more of these positions; and antibodies comprisingthree or more modified amino acid residues at three or more of thesepositions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H019, H025, H040, H052, H070,and H110; and EU residues corresponding to H119, H121, H136, H180, H190,H221, H222, and H404 (see Table 2, “SSHC-11” and the correspondingpositions of the representative heavy chain polypeptide according to SEQID NO:1). Specifically provided herein, are antibodies comprising one ormore modified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H005, H023, H042, H065, H074,and H084; and EU residues corresponding to 1-1118, H119, H132, H134,H135, H136, H137, H138, H139, H155, H160, H162, H165, H172, H174, H176,H177, H191, H194, H219, H238, H239, H241, H243, H246, H262, H264, H265,H267, H268, H269, H270, H271; H272, H274, H275, H278, H280, H281, H282,H283, H286, H289, H292, H293, H294, H295, H296, H297, H298, H299, H300,H301, H303, H305, H317, H320, H324, H326, H327, H329, H330, H332, H333,H334, H335, H337, H339, H340, H342, H344, H355, H356, H358, H359, H360,H375, H383, H384, H386, H389, H392, H398, H404, H420, H421, H436, andH438 (see Table 2, “SSHC-12” and the corresponding positions of therepresentative heavy chain polypeptide according to SEQ ID NO:1).Specifically provided herein are antibodies comprising one or moremodified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H005, H023, H074, and H084;and EU residues corresponding to H118, H119, H132, H134, H135, H136,H137, H139, H160, H162, H165, H172, H191, H194, H239, H241, H246, H267,H268, H269, H270, H271, H272, H274, H275, H280, H281, H282, H283, H286,H289, H292, H293, H294, H295, H296, H297, H298, H299, H300, H301, H303,H305, H317, H320, H324, H326, H327, H329, H330, H332, H333, H334, H335,H337, H339, H340, H342, H344, H355, H359, H375, H386, H389, H392, H398,H404, H420, H421, and H438 (see Table 2, “SSHC-13” and the correspondingpositions of the representative heavy chain polypeptide according to SEQID NO:1). Specifically provided herein are antibodies comprising one ormore modified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H023 and H074; and EUresidues corresponding to H119, H134, H135, H137, H139, H160, H162,H165, H172, H191, H194, H241, H246, H267, H268, H269, H270, H271, H272,H274, H275, H280, H281, H282, H283, H286, H289, H292, H294, H295, H296,H297, H298, H299, H300, H301, H303, H305, H317, H320, H324, H326, H327,H329, H330, H332, H333, H335, H337, H339, H340, H342, H344, H355, H375,H386, H392, H398, H404, H420, H421, and H438 (see Table 2, “SSHC-14” andthe corresponding positions of the representative heavy chainpolypeptide according to SEQ ID NO:1). Specifically provided herein areantibodies comprising one or more modified amino acid residues at one ormore of these positions; antibodies comprising one or more modifiedamino acid residues at two or more of these positions; antibodiescomprising one or more modified amino acid residues at three or more ofthese positions; antibodies comprising two or more modified amino acidresidues at one or more of these positions; antibodies comprising two ormore modified amino acid residues at two or more of these positions;antibodies comprising two or more modified amino acid residues at threeor more of these positions; antibodies comprising three or more modifiedamino acid residues at one or more of these positions; antibodiescomprising three or more modified amino acid residues at two or more ofthese positions; and antibodies comprising three or more modified aminoacid residues at three or more of these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H042 and H065; and EUresidues corresponding to H138, H155, H174, H176, H177, H219, H238,H243, H262, H264, H265, H278, H342, H356, H358, H360, H383, H404, H384and H436 (see Table 2, “SSHC-15” and the corresponding positions of therepresentative heavy chain polypeptide according to SEQ ID NO:1).Specifically provided herein are antibodies comprising one or moremodified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H005 and H084; and EUresidues corresponding to H118, H132, H136, H239, H293, H334, H342,H355, H359, H389, and H404 (see Table 2, “SSHC-16” and the correspondingpositions of the representative heavy chain polypeptide according to SEQID NO:1). Specifically provided herein are antibodies comprising one ormore modified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H005, H023, and H084; and EUresidues corresponding to H118, H119, H132, H134, H136, H137, H160,H162, H172, H239, H241, H267, H269, H270, H271, H272, H282, H286, H292,H293, H296, H298, H329, H330, H334, H335, H340, H342, H355, H359, H386,H389, H404, H420, H421, and H438 (see Table 2, “SSHC-17” and thecorresponding positions of the representative heavy chain polypeptideaccording to SEQ ID NO:1). Specifically provided herein are antibodiescomprising one or more modified amino acid residues at one or more ofthese positions; antibodies comprising one or more modified amino acidresidues at two or more of these positions; antibodies comprising one ormore modified amino acid residues at three or more of these positions;antibodies comprising two or more modified amino acid residues at one ormore of these positions; antibodies comprising two or more modifiedamino acid residues at two or more of these positions; antibodiescomprising two or more modified amino acid residues at three or more ofthese positions; antibodies comprising three or more modified amino acidresidues at one or more of these positions; antibodies comprising threeor more modified amino acid residues at two or more of these positions;and antibodies comprising three or more modified amino acid residues atthree or more of these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H023 and H084; and EUresidues corresponding to H118, H119, H135, H136, H137, H160, H161,H162, H164, H195, H197, H219, H282, H289, H296, H330, H335, H361, H400,H404, H422, H440, H260, H267, H268, H272, H274, H292, H293, H297, H298,H303, H305, H332, H333, H334, H340, H341, H342, H343, H355, H362, H386,H392, H404, H424, H438, H442 and H443 (see Table 2, “SSHC-18” and thecorresponding positions of the representative heavy chain polypeptideaccording to SEQ ID NO:1). Specifically provided herein are antibodiescomprising one or more modified amino acid residues at one or more ofthese positions; antibodies comprising one or more modified amino acidresidues at two or more of these positions; antibodies comprising one ormore modified amino acid residues at three or more of these positions;antibodies comprising two or more modified amino acid residues at one ormore of these positions; antibodies comprising two or more modifiedamino acid residues at two or more of these positions; antibodiescomprising two or more modified amino acid residues at three or more ofthese positions; antibodies comprising three or more modified amino acidresidues at one or more of these positions; antibodies comprising threeor more modified amino acid residues at two or more of these positions;and antibodies comprising three or more modified amino acid residues atthree or more of these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H023 and H084; and EUresidues corresponding to H118, H119, H135, H116, H137, H160, H161,H162, H164, H195, H197, H219, H282, H296, H335, H361, H422, H440, H267,H272, H274, H293, H297, H298, H303, H305, H334, H340, H341, H342, H343,H355, H362, H392, H404, H424, H438, H442 and H443 (see Table 2,“SSHC-19” and the corresponding positions of the representative heavychain polypeptide according to SEQ ID NO:1). Specifically providedherein are antibodies comprising one or more modified amino acidresidues at one or more of these positions; antibodies comprising one ormore modified amino acid residues at two or more of these positions;antibodies comprising one or more modified amino acid residues at threeor more of these positions; antibodies comprising two or more modifiedamino acid residues at one or more of these positions; antibodiescomprising two or more modified amino acid residues at two or more ofthese positions; antibodies comprising two or more modified amino acidresidues at three or more of these positions; antibodies comprisingthree or more modified amino acid residues at one or more of thesepositions; antibodies comprising three or more modified amino acidresidues at two or more of these positions; and antibodies comprisingthree or more modified amino acid residues at three or more of thesepositions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H023 and H084; and EUresidues corresponding to H118, H119, H135, H136, H137, H160, H161,H162, H195, H197, H219, H282, H296, H422, H440, H267, H272, H293, H297,H298, H303, H305, H334, H340, H341, H342, H355, H392, H404, H424, H438,H442 and H443 (see Table 2, “SSHC-20” and the corresponding positions ofthe representative heavy chain polypeptide according to SEQ ID NO:1).Specifically provided herein are antibodies comprising one or moremodified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H023 and H084; and EUresidues corresponding to H118, H119, H135, H136, H160, H162, H195,H219, H282, H296, H267, H293, H297, H298, H303, H305, H334, H340, H341,H392, H438, and H442 (see Table 2, “SSHC-21” and the correspondingpositions of the representative heavy chain polypeptide according to SEQID NO:1). Specifically provided herein are antibodies comprising one ormore modified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H003, H007, H014, H016, H019,H025, H040, H042, H043, H051, H052, H053, H056, H070, H082A, H098, H100,H110, and H112; and EU residues corresponding to H121, H180, H184, H190,H192, H214, H216, H221, H222, H225, H227, H230, H231, H232, and H236(see Table 2, “SSHC-22” and the corresponding positions of therepresentative heavy chain polypeptide according to SEQ ID NO:1).Specifically provided herein are antibodies comprising one or moremodified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H003, H007, H014, H016, H019,H025, H040, H042, H043, H052, H053, H056, H070, H082A, H100, H110, andH112; and EU residues corresponding to H121, H180, H190, H192, H214,H216, H221, H222, H225, H227, H230, H231, H232, and H236 (see Table 2,“SSHC-23” and the corresponding positions of the representative heavychain polypeptide according to SEQ ID NO:1). Specifically providedherein are antibodies comprising one or more modified amino acidresidues at one or more of these positions; antibodies comprising one ormore modified amino acid residues at two or more of these positions;antibodies comprising one or more modified amino acid residues at threeor more of these positions; antibodies comprising two or more modifiedamino acid residues at one or more of these positions; antibodiescomprising two or more modified amino acid residues at two or more ofthese positions; antibodies comprising two or more modified amino acidresidues at three or more of these positions; antibodies comprisingthree or more modified amino acid residues at one or more of thesepositions; antibodies comprising three or more modified amino acidresidues at two or more of these positions; and antibodies comprisingthree or more modified amino acid residues at three or more of thesepositions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H003, H007, H014, H016, H019,H025, H040, H042, H043, H052, H070, H082A, H100, H110, and H112; and EUresidues corresponding to H121, H180, H190, H192, H214, H216, H221,H222, H225, H227, H230, H231, H232, and H236 (see Table 2, “SSHC-24” andthe corresponding positions of the representative heavy chainpolypeptide according to SEQ ID NO:1). Specifically provided herein areantibodies comprising one or more modified amino acid residues at one ormore of these positions; antibodies comprising one or more modifiedamino acid residues at two or more of these positions; antibodiescomprising one or more modified amino acid residues at three or more ofthese positions; antibodies comprising two or more modified amino acidresidues at one or more of these positions; antibodies comprising two ormore modified amino acid residues at two or more of these positions;antibodies comprising two or more modified amino acid residues at threeor more of these positions; antibodies comprising three or more modifiedamino acid residues at one or more of these positions; antibodiescomprising three or more modified amino acid residues at two or more ofthese positions; and antibodies comprising three or more modified aminoacid residues at three or more of these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H007, H014, H019, H025, H040,H043, H052, H070, H100, H110, and H112; and EU residues corresponding toH121, H180, H214, H216, H222, H227, H230, H231, H232, and H236 (seeTable 2, “SSHC-25” and the corresponding positions of the representativeheavy chain polypeptide according to SEQ ID NO:1). Specifically providedherein are antibodies comprising one or more modified amino acidresidues at one or more of these positions; antibodies comprising one ormore modified amino acid residues at two or more of these positions;antibodies comprising one or more modified amino acid residues at threeor more of these positions; antibodies comprising two or more modifiedamino acid residues at one or more of these positions; antibodiescomprising two or more modified amino acid residues at two or more ofthese positions; antibodies comprising two or more modified amino acidresidues at three or more of these positions; antibodies comprisingthree or more modified amino acid residues at one or more of thesepositions; antibodies comprising three or more modified amino acidresidues at two or more of these positions; and antibodies comprisingthree or more modified amino acid residues at three or more of thesepositions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H019, H025, H051, H070, H098,H110, and H112; and EU residues corresponding to H121, H136, H180, H187,H190, H214, H216, H221, and H222 (see Table 2, “SSHC-26” and thecorresponding positions of the representative heavy chain polypeptideaccording to SEQ ID NO:1). Specifically provided herein are antibodiescomprising one or more modified amino acid residues at one or more ofthese positions; antibodies comprising one or more modified amino acidresidues at two or more of these positions; antibodies comprising one ormore modified amino acid residues at three or more of these positions;antibodies comprising two or more modified amino acid residues at one ormore of these positions; antibodies comprising two or more modifiedamino acid residues at two or more of these positions; antibodiescomprising two or more modified amino acid residues at three or more ofthese positions; antibodies comprising three or more modified amino acidresidues at one or more of these positions; antibodies comprising threeor more modified amino acid residues at two or more of these positions;and antibodies comprising three or more modified amino acid residues atthree or more of these positions.

In certain embodiments, provided herein are antibodies comprising one ormore modified amino acid residues at one or more positions selected fromKabat or Chothia residues corresponding to H019, H025, H051, H070, H077,H079, H098, H110, and H112; and EU residues corresponding to H121, H136,H180, H187, H190, H214, H216, H221, and H222 (see Table 2, “SSHC-27” andthe corresponding positions of the representative heavy chainpolypeptide according to SEQ ID NO:1). Specifically provided herein areantibodies comprising one or more modified amino acid residues at one ormore of these positions; antibodies comprising one or more modifiedamino acid residues at two or more of these positions; antibodiescomprising one or more modified amino acid residues at three or more ofthese positions; antibodies comprising two or more modified amino acidresidues at one or more of these positions; antibodies comprising two ormore modified amino acid residues at two or more of these positions;antibodies comprising two or more modified amino acid residues at threeor more of these positions; antibodies comprising three or more modifiedamino acid residues at one or more of these positions; antibodiescomprising three or more modified amino acid residues at two or more ofthese positions; and antibodies comprising three or more modified aminoacid residues at three or more of these positions.

Examples of Site-Specific Positions for Substitution: Light Chains

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues L007 and L022; and EU light chainresidue L152 (see Table 3, “SSLC-1” and the corresponding positions ofthe representative light chain polypeptide according to SEQ 1D NO:2).Specifically provided herein are antibodies comprising one or moremodified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues L043, L049, L056, L057, L060,L067, and L068; and EU light chain residues L109, L112, L114, L144,L153, L156, L157, L168, L184, L202, L203, and L206 (see Table 3,“SSLC-2” and the corresponding positions of the representative lightchain polypeptide according to SEQ ID NO:2). Specifically providedherein are antibodies comprising one or more modified amino acidresidues at one or more of these positions; antibodies comprising one ormore modified amino acid residues at two or more of these positions;antibodies comprising one or more modified amino acid residues at threeor more of these positions; antibodies comprising two or more modifiedamino acid residues at one or more of these positions; antibodiescomprising two or more modified amino acid residues at two or more ofthese positions; antibodies comprising two or more modified amino acidresidues at three or more of these positions; antibodies comprisingthree or more modified amino acid residues at one or more of thesepositions; antibodies comprising three or more modified amino acidresidues at two or more of these positions; and antibodies comprisingthree or more modified amino acid residues at three or more of thesepositions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues L043, L049, L056, L057, L060,L067, and L068; and EU light chain residues L109, L112, L114, L144,L153, L156, L168, L184, L202, and L203 (see Table 3, “SSLC-3” and thecorresponding positions of the representative light chain polypeptideaccording to SEQ ID NO:2). Specifically provided herein are antibodiescomprising one or more modified amino acid residues at one or more ofthese positions; antibodies comprising one or more modified amino acidresidues at two or more of these positions; antibodies comprising one ormore modified amino acid residues at three or more of these positions;antibodies comprising two or more modified amino acid residues at one ormore of these positions; antibodies comprising two or more modifiedamino acid residues at two or more of these positions; antibodiescomprising two or more modified amino acid residues at three or more ofthese positions; antibodies comprising three or more modified amino acidresidues at one or more of these positions; antibodies comprising threeor more modified amino acid iesidues al two or more of these positions;and antibodies comprising three or more modified amino acid residues atthree or more of these positions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues L043, L049, L056, L057, L060,L067, and L068; and EU light chain residues L109, L144, L153, L156,L184, L202, and L203 (see Table 3, “SSLC-4” and the correspondingpositions of the representative light chain polypeptide according to SEQID NO:2). Specifically provided herein are antibodies comprising one ormore modified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues L049, L056, L057, L060, and L067;and EU light chain residues L109, L153, L202, and L203 (see Table 3,“SSLC-5” and the corresponding positions of the representative lightchain polypeptide according to SEQ ID NO:2). Specifically providedherein are antibodies comprising one or more modified amino acidresidues at one or more of these positions; antibodies comprising one ormore modified amino acid residues at two or more of these positions;antibodies comprising one or more modified amino acid residues at threeor more of these positions; antibodies comprising two or more modifiedamino acid residues at one or more of these positions; antibodiescomprising two or more modified amino acid residues at two or more ofthese positions; antibodies comprising two or more modified amino acidresidues at three or more of these positions; antibodies comprisingthree or more modified amino acid residues at one or more of thesepositions; antibodies comprising three or more modified amino acidresidues at two or more of these positions; and antibodies comprisingthree or more modified amino acid residues at three or more of thesepositions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues minus 1, L003, L005, L007, L008,L009, L010, L016, L017, L018, L020, L022, L026, L027, L045, L058, L063,L065, L066, L070, L077, L079, and L107; and EU light chain residuesL138, L142, L143, L152, L171, L182, L188, L199, and L201 (see Table 3,“SSLC-6” and the corresponding positions of the representative lightchain polypeptide according to SEQ ID NO:2). Specifically providedherein are antibodies comprising one or more modified amino acidresidues at one or more of these positions; antibodies comprising one ormore modified amino acid residues at two or more of these positions;antibodies comprising one or more modified amino acid residues at threeor more of these positions; antibodies comprising two or more modifiedamino acid residues at one or more of these positions; antibodiescomprising two or more modified amino acid residues at two or more ofthese positions; antibodies comprising two or more modified amino acidresidues at three or more of these positions; antibodies comprisingthree or more modified amino acid residues at one or more of thesepositions; antibodies comprising three or more modified amino acidresidues at two or more of these positions; and antibodies comprisingthree or more modified amino acid residues at three or more of thesepositions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues minus 1, L003, L005, L007, L008,L009, L010, L016, L017, L018, L020, L022, L026, L027, L045, L058, L063,L065, L066, L070, L077, L079, and L107; and EU light chain residuesL142, L143, L152, L171, L182, L188, L199, and L201 (see Table 3,“SSLC-7” and the corresponding positions of the representative lightchain polypeptide according to SEQ ID NO:2). Specifically providedherein are antibodies comprising one or more modified amino acidresidues at one or more of these positions; antibodies comprising one ormore modified amino acid residues at two or more of these positions;antibodies comprising one or more modified amino acid residues at threeor more of these positions; antibodies comprising two or more modifiedamino acid residues at one or more of these positions; antibodiescomprising two or more modified amino acid residues at two or more ofthese positions; antibodies comprising two or more modified amino acidresidues at three or more of these positions; antibodies comprisingthree or more modified amino acid residues at one or more of thesepositions; antibodies comprising three or more modified amino acidresidues at two or more of these positions; and antibodies comprisingthree or more modified amino acid residues at three or more of thesepositions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues minus 1, L003, L005, L007, L008,L009, L016, L017, L018, L020, L022, L026, L027, L045, L058, L063, L065,L066, L070, L077, L079, and L107; and EU light chain residues L142,L152, L171, L182, L188, and L199 (see Table 3, “SSLC-8” and thecorresponding positions of the representative light chain polypeptideaccording to SEQ ID NO:2). Specifically provided herein are antibodiescomprising one or more modified amino acid residues at one or more ofthese positions; antibodies comprising one or more modified amino acidresidues at two or more of these positions; antibodies comprising one ormore modified amino acid residues at three or more of these positions;antibodies comprising two or more modified amino acid residues at one ormore of these positions; antibodies comprising two or more modifiedamino acid residues at two or more of these positions; antibodiescomprising two or more modified amino acid residues at three or more ofthese positions; antibodies comprising three or more modified amino acidresidues at one or more of these positions; antibodies comprising threeor more modified amino acid residues at two or more of these positions;and antibodies comprising three or more modified amino acid residues atthree or more of these positions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues minus 1, L005, L007, L008, L016,L017, L018, L020, L022, L027, L045, L058, L063, L077, L079, and L107;and EU light chain residues L142, L152, L182, L188, and L199 (see Table3, “SSLC-9” and the corresponding positions of the representative lightchain polypeptide according to SEQ ID NO:2). Specifically providedherein are antibodies comprising one or more modified amino acidresidues at one or more of these positions; antibodies comprising one ormore modified amino acid residues at two or more of these positions;antibodies comprising one or more modified amino acid residues at threeor more of these positions; antibodies comprising two or more modifiedamino acid residues at one or more of these positions; antibodiescomprising two or more modified amino acid residues at two or more ofthese positions; antibodies comprising two or more modified amino acidresidues at three or more of these positions; antibodies comprisingthree or more modified amino acid residues at one or more of thesepositions; antibodies comprising three or more modified amino acidresidues at two or more of these positions; and antibodies comprisingthree or more modified amino acid residues at three or more of thesepositions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues minus 1, L016, and L063; and EUlight chain residue L199 (see Table 3, “SSLC-10” and the correspondingpositions of the representative light chain polypeptide according to SEQID NO:2). Specifically provided herein are antibodies comprising one ormore modified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues minus 1, L007, L008, L014, L016,L022, L063, and L070; and EU light chain residues L138, L142, L143 andL152 (see Table 3, “SSLC-11” and the corresponding positions of therepresentative light chain polypeptide according to SEQ ID NO:2).Specifically provided herein are antibodies comprising one or moremodified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues minus 1, L007, L008, L016, L022,L063, and L070; and EU light chain residues L138, L142, L143, L152 andL201 (see Table 3, “SSLC-12” and the corresponding positions of therepresentative light chain polypeptide according to SEQ ID NO:2).Specifically provided herein are antibodies comprising one or moremodified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues minus 1, L003, L005, L007, L008,L009, L010, L016, L017, L018, L020, L022, L026, L027, L045, L058, L063,L065, L066, L070, L077, L079, and L107; and EU light chain residuesL138, L142, L143, L152, L171, L182, L188, L199, and L201 (see Table 3,“SSLC-13” and the corresponding positions of the representative lightchain polypeptide according to SEQ ID NO:2). Specifically providedherein are antibodies comprising one or more modified amino acidresidues at one or more of these positions; antibodies comprising one ormore modified amino acid residues at two or more of these positions;antibodies comprising one or more modified amino acid residues at threeor more of these positions; antibodies comprising two or more modifiedamino acid residues at one or more of these positions; antibodiescomprising two or more modified amino acid residues at two or more ofthese positions; antibodies comprising two or more modified amino acidresidues at three or more of these positions; antibodies comprisingthree or more modified amino acid residues at one or more of thesepositions; antibodies comprising three or more modified amino acidresidues at two or more of these positions; and antibodies comprisingthree or more modified amino acid residues at three or more of thesepositions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues minus 1, L003, L005, L007, L008,LOO 9, L016, L017, L018, L020, L022, L026, L027, L045, L058, L063, L065,L066, L070, L077, L079, and L107; and EU light chain residues L142,L152, L171, L182, L188, and L199 (see Table 3, “SSLC-14” and thecorresponding positions of the representative light chain polypeptideaccording to SEQ ID NO:2). Specifically provided herein are antibodiescomprising one or more modified amino acid residues at one or more ofthese positions; antibodies comprising one or more modified amino acidresidues at two or more of these positions; antibodies comprising one ormore modified amino acid residues at three or more of these positions;antibodies comprising two or more modified amino acid residues at one ormore of these positions; antibodies comprising two or more modifiedamino acid residues at two or more of these positions; antibodiescomprising two or more modified amino acid residues at three or more ofthese positions; antibodies comprising three or more modified amino acidresidues at one or more of these positions; antibodies comprising threeor more modified amino acid residues at two or more of these positions;and antibodies comprising three or more modified amino acid residues atthree or more of these positions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues minus 1, L005, L007, L008, L016,L017, L018, L020, L022, L027, L045, L058, L063, L077, L079, and L107;and EU light chain residues L142, L152, L182, L188, and L199 (see Table3, “SSLC-15” and the corresponding positions of the representative lightchain polypeptide according to SEQ ID NO:2). Specifically providedherein are antibodies comprising one or more modified amino acidresidues at one or more of these positions; antibodies comprising one ormore modified amino acid residues at two or more of these positions;antibodies comprising one or more modified amino acid residues at threeor more of these positions; antibodies comprising two or more modifiedamino acid residues at one or more of these positions; antibodiescomprising two or more modified amino acid residues at two or more ofthese positions; antibodies comprising two or more modified amino acidresidues at three or more of these positions; antibodies comprisingthree or more modified amino acid residues at one or more of thesepositions; antibodies comprising three or more modified amino acidresidues at two or more of these positions; and antibodies comprisingthree or more modified amino acid residues at three or more of thesepositions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues minus 1, L016 and L063; and EUlight chain residue L199 (see Table 3, “SSLC-16” and the correspondingpositions of the representative light chain polypeptide according to SEQID NO:2). Specifically provided herein are antibodies comprising one ormore modified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues minus 1, L007, L008, L014, L016,L022, L063, and L070; and EU light chain residues L138, L142, L143 andL152 (see Table 3, “SSLC-17” and the corresponding positions of therepresentative light chain polypeptide according to SEQ ID NO:2).Specifically provided herein are antibodies comprising one or moremodified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues minus 1, L007, L008, L016, 22,L063, and L070; and EU light chain residues L138, L142, L143, L152, andL201 (see Table 3, “SSLC-18” and the corresponding positions of therepresentative light chain polypeptide according to SEQ ID NO:2).Specifically provided herein are antibodies comprising one or moremodified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues L043, L049, L056, L057, L060,L067, and L068; and EU light chain residues L109, L112, L114, L144,L153, L156, L157, L168, L184, L202, L203, and L206 (see Table 3,“SSLC-19” and the corresponding positions of the representative lightchain polypeptide according to SEQ ID NO:2). Specifically providedherein are antibodies comprising one or more modified amino acidresidues at one or more of these positions; antibodies comprising one ormore modified amino acid residues at two or more of these positions;antibodies comprising one or more modified amino acid residues at threeor more of these positions; antibodies comprising two or more modifiedamino acid residues at one or more of these positions; antibodiescomprising two or more modified amino acid residues at two or more ofthese positions; antibodies comprising two or more modified amino acidresidues at three or more of these positions; antibodies comprisingthree or more modified amino acid residues at one or more of thesepositions; antibodies comprising three or more modified amino acidresidues at two or more of these positions; and antibodies comprisingthree or more modified amino acid residues at three or more of thesepositions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues L043, L049, L056, L057, L060,L067, and L068; and EU light chain residues L109, L112, L144, L153,L156, L168, L184, L202, and L203 (see Table 3, “SSLC-20” and thecorresponding positions of the representative light chain polypeptideaccording to SEQ ID NO:2). Specifically provided herein are antibodiescomprising one or more modified amino acid residues at one or more ofthese positions; antibodies comprising one or more modified amino acidresidues at two or more of these positions; antibodies comprising one ormore modified amino acid residues at three or more of these positions;antibodies comprising two or more modified amino acid residues at one ormore of these positions; antibodies comprising two or more modifiedamino acid residues at two or more of these positions; antibodiescomprising two or more modified amino acid residues at three or more ofthese positions; antibodies comprising three or more modified amino acidresidues at one or more of these positions; antibodies comprising threeor more modified amino acid residues at two or more of these positions;and antibodies comprising three or more modified amino acid residues atthree or more of these positions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues L043, L049, L056, L057, L060,L067, and L068; and EU light chain residues L109, L144, L153, L156,L184, L202, and L203 (see Table 3, “SSLC-21” and the correspondingpositions of the representative light chain polypeptide according to SEQID NO:2). Specifically provided herein are antibodies comprising one ormore modified amino acid residues at one or more of these positions;antibodies comprising one or more modified amino acid residues at two ormore of these positions; antibodies comprising one or more modifiedamino acid residues at three or more of these positions; antibodiescomprising two or more modified amino acid residues at one or more ofthese positions; antibodies comprising two or more modified amino acidresidues at two or more of these positions; antibodies comprising two ormore modified amino acid residues at three or more of these positions;antibodies comprising three or more modified amino acid residues at oneor more of these positions; antibodies comprising three or more modifiedamino acid residues at two or more of these positions; and antibodiescomprising three or more modified amino acid residues at three or moreof these positions.

In certain embodiments, the site-specific positions are selected fromKabat or Chothia light chain residues L049, L056, L057, L060, and L067;and EU light chain residues L109, L153, L202, and L203 (see Table 3,“SSLC-22” and the corresponding positions of the representative lightchain polypeptide according to SEQ ID NO:2). Specifically providedherein are antibodies comprising one or more modified amino acidresidues at one or more of these positions; antibodies comprising one ormore modified amino acid residues at two or more of these positions;antibodies comprising one or more modified amino acid residues at threeor more of these positions; antibodies comprising two or more modifiedamino acid residues at one or more of these positions; antibodiescomprising two or more modified amino acid residues at two or more ofthese positions; antibodies comprising two or more modified amino acidresidues at three or more of these positions; antibodies comprisingthree or more modified amino acid residues at one or more of thesepositions; antibodies comprising three or more modified amino acidresidues at two or more of these positions; and antibodies comprisingthree or more modified amino acid residues at three or more of thesepositions.

Examples of Site-Specific Positions for Substitution: Heavy and LightChains

Specifically contemplated is every site-specific heavy chainmodification described herein with every site-specific light chainmodification described herein.

In some embodiments, the site-specific positions are selected from Kabator Chothia heavy chain residues H019, H025, H040, H052, H071, and H117;and EU heavy chain residues H119, H124, H139, H183, H193, H224, H225,and H407 (see Table 2, “SSHC-7” and the corresponding positions of therepresentative heavy chain polypeptide according to SEQ ID NO:1) andKabat or Chothia light chain residues L007 and L022; and EU light chainresidue L152 (see Table 3, “SSLC-1” and the corresponding positions ofthe representative light chain polypeptide according to SEQ ID NO:2). Insome embodiments, at least one of these positions is modified in theheavy chain and at least one of these positions is modified in the lightchain. In some embodiments, at least two of these positions are modifiedin the heavy chain and at least one of these positions is modified inthe light chain. In some embodiments, at least one of these positions ismodified in the heavy chain and at least two of these positions aremodified in the light chain.

In some embodiments, the site-specific positions are selected from Kabator Chothia heavy chain residues H019, H025, H040, H052, and H070; and EUheavy chain residues H119, H121, H136, H180, H190, H222, H241, and H404(see Table 2, “SSHC-8” and the corresponding positions of therepresentative heavy chain polypeptide according to SEQ ID NO:1) andKabat or Chothia light chain residues L007 and L022; and EU light chainresidue L152 (see Table 3, “SSLC-1” and the corresponding positions ofthe representative light chain polypeptide according to SEQ ID NO:2). Insome embodiments, at least one of these positions is modified in theheavy chain and at least one of these positions is modified in the lightchain. In some embodiments, at least two of these positions are modifiedin the heavy chain and at least one of these positions is modified inthe light chain. In some embodiments, at least one of these positions ismodified in the heavy chain and at least two of these positions aremodified in the light chain.

In some embodiments, the site-specific positions are selected from Kabator Chothia heavy chain residues H025 and H040; and EU heavy chainresidues H119, H121, H136, H180, H190, H222, H241, and H404 (see Table2, “SSHC-9” and the corresponding positions of the representative heavychain polypeptide according to SEQ ID NO:1) and Kabat or Chothia lightchain residues L007 and L022; and EU light chain residue L152 (see Table3, “SSLC-1” and the corresponding positions of the representative lightchain polypeptide according to SEQ ID NO:2). In some embodiments, atleast one of these positions is modified in the heavy chain and at leastone of these positions is modified in the light chain. In someembodiments, at least two of these positions are modified in the heavychain and at least one of these positions is modified in the lightchain. In some embodiments, at least one of these positions is modifiedin the heavy chain and at least two of these positions are modified inthe light chain.

In some embodiments, the site-specific positions are selected from EUheavy chain residues H121, H136, H180, H241, and H404 (see Table 2,“SSHC-10” and the corresponding positions of the representative heavychain polypeptide according to SEQ ID NO:1) and Kabat or Chothia lightchain residues L007 and L022; and EU light chain residue L152 (see Table3, “SSLC-1” and the corresponding positions of the representative lightchain polypeptide according to SEQ ID NO:2). In some embodiments, atleast one of these positions is modified in the heavy chain and at leastone of these positions is modified in the light chain. In someembodiments, at least two of these positions are modified in the heavychain and at least one of these positions is modified in the lightchain. In some embodiments, at least one of these positions is modifiedin the heavy chain and at least two of these positions are modified inthe light chain.

In some embodiments, the site-specific positions are selected from Kabator Chothia heavy chain residues H019, H025, H040, H052, H070, and H110;and EU heavy chain residues H119, H121, H136, H180, H190, H221, H222,and H404 (see Table 2, “SSHC-11” and the corresponding positions of therepresentative heavy chain polypeptide according to SEQ ID NO:1) andKabat or Chothia light chain residues L007 and L022; and EU light chainresidue L152 (see Table 3, “SSLC-1” and the corresponding positions ofthe representative light chain polypeptide according to SEQ ID NO:2). Insome embodiments, at least one of these positions is modified in theheavy chain and at least one of these positions is modified in the lightchain. In some embodiments, at least two of these positions are modifiedin the heavy chain and at least one of these positions is modified inthe light chain. In some embodiments, at least one of these positions ismodified in the heavy chain and at least two of these positions aremodified in the light chain.

In certain embodiments, provided herein are antibodies comprising apolypeptide chain having at least 70%, 80%, 90%, 95%, or 99% homology toSEQ ID NO:1 and having one or more modified amino acid residues at sitesselected from Kabat or Chothia heavy chain residues H019, H025, H040,H052, and H070; and EU heavy chain residues H119, H121, H136, H180,H190, H222, H241, and H404 (see Table 2, “SSHC-8”). Specificallyprovided herein are antibodies comprising one or more modified aminoacid residues at one or more of these positions; antibodies comprisingone or more modified amino acid residues at two or more of thesepositions; antibodies comprising one or more modified amino acidresidues at three or more of these positions; antibodies comprising twoor more modified amino acid residues at one or more of these positions;antibodies comprising two or more modified amino acid residues at two ormore of these positions; antibodies comprising two or more modifiedamino acid residues at three or more of these positions; antibodiescomprising three or more modified amino acid residues at one or more ofthese positions; antibodies comprising three or more modified amino acidresidues at two or more of these positions; and antibodies comprisingthree or more modified amino acid residues at three or more of thesepositions.

In certain embodiments, provided herein are antibodies comprising apolypeptide chain having at least 70%, 80%, 90%, 95%, or 99% homology toSEQ ID NO:2 and having one or more modified amino acid residues at sitesselected from Kabat or Chothia light chain residues L007 and L022; andEU light chain residue L152 (see Table 3, “SSLC-1”). Specificallyprovided herein are antibodies comprising one or more modified aminoacid residues at one or more of these positions; antibodies comprisingone or more modified amino acid residues at two or more of thesepositions; antibodies comprising one or more modified amino acidresidues at all three of these positions; antibodies comprising two ormore modified amino acid residues at one or more of these positions;antibodies comprising two or more modified amino acid residues at two Ormore of these positions; antibodies comprising two or more modifiedamino acid residues at all three of these positions; antibodiescomprising three modified amino acid residues at one or more of thesepositions; antibodies comprising three modified amino acid residues attwo or more of these positions; and antibodies comprising three modifiedamino acid residues at all three of these positions.

In certain embodiments, each modified amino acid residue isindependently at a specific site selected from the group consisting ofoptimally substitutable positions of any polypeptide chain of theantibody. In certain embodiments, the antibodies comprise two or moresite-specific modified amino acid residues in a single light chainpolypeptide. In certain embodiments, the antibodies comprise two or moresite-specific modified amino acid residues in a single heavy chainpolypeptide. In certain embodiments, the antibodies comprise at leastone site-specific modified amino acid residue in a light chainpolypeptide and at least one site-specific modified amino acid residuein a heavy chain polypeptide.

In certain embodiments, the antibodies comprise at least onesite-specific modified amino acid residue in a light chain polypeptideand at least one site-specific modified amino acid residue in each oftwo heavy chain polypeptides. In certain embodiments, the antibodiescomprise at least one site-specific modified amino acid residue in eachof two light chain polypeptides and at least one site-specific modifiedamino acid residue in a heavy chain polypeptide. In certain embodiments,the antibodies comprise at least one site-specific modified amino acidresidue in each of two light chain polypeptides and at least onesite-specific modified amino acid residue in each of two heavy chainpolypeptides.

In certain embodiments, the antibodies comprise three or more, four ormore, five or more, or six or more site-specific modified amino acidresidues. In certain embodiments, the antibodies comprise two to sixmodified amino acid residues. In certain embodiments, the antibodiescomprise three to six modified amino acid residues. In certainembodiments, the antibodies comprise four to six modified amino acidresidues. In certain embodiments, the antibodies comprise five to sixmodified amino acid residues.

The antibody can have any form recognized by those of skill in the art.The antibody can comprise a single polypeptide chain—a single heavychain or a single light chain. The antibody can also form multimers thatwill be recognized by those of skill in the art including homodimers,heterodimers, homomultimers, and heteromultimers. These multimers can belinked or unlinked. Useful linkages include interchain disulfide bondstypical for polypeptide molecules. The multimers can also be linked byother amino acid residues, including modified amino acid residuesderived from the modified amino acids described herein. The antibody canbe an immunoglobulin such as of any class or subclass including IgA,IgA1, IgA2, IgD, IgE, IgG, IgG1, IgG2, IgG3, IgG4 and IgM. The antibodycan be of the form of any antibody fragment including Fv, Fc, Fab, and(Fab′)₂ and scFv.

A parent antibody can have affinity to any antigen known to those ofskill in the art, or later discovered. Virtually any substance may be anantigen for a parent antibody, or an antibody of the presentdescription. Examples of useful antigens include, but are not limitedto, alpha-1 antitrypsin, angiostatin, antihemolytic factor,polypeptides, apolipoprotein, apoprotein, atrial natriuretic factor,atrial natriuretic polypeptide, atrial peptides, C—X—C chemokines (e.g.,T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1,PF4, MIG), calcitonin, CC chemokines (e.g., monocyte chemoattractantprotein-1, monocyte chemoattractant protein-2, monocyte chemoattractantprotein-3, monocyte inflammatory protein-1 alpha, monocyte inflammatoryprotein-1 beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065,T64262), CD40 ligand, C-kit ligand, collagen, colony stimulating factor(CSF), complement factor 5a, complement inhibitor, complement receptor1, cytokines, (e.g., epithelial neutrophil activating peptide-78,GRO/MGSA, GRO, GRO, MIP-1, MIP-1, MCP-1), epidermal growth factor (EGF),erythropoietin (“EPO”), exfoliating toxins A and B, factor IX, factorVII, factor VIII, factor X, fibroblast growth factor (FGF), fibrinogen,fibronectin, G-CSF, GM-CSF, glucocerebrosidase, gonadotropin, growthfactors, hedgehog proteins (e.g., Sonic, Indian, Desert), hemoglobin,hepatocyte growth factor (HGF), hirudin, human serum albumin, insulin,insulin-like growth factor (IGF), interferons (e.g., IFN-α, IFN-,IFN-γ), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, etc.), keratinocyte growth factor(KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin,neutrophil inhibitory factor (NIF), oncostatin M, osteogenic protein,parathyroid hormone, PD-ECSF, PDGF, peptide hormones (e.g., human growthhormone), pleiotropin, protein A, protein G, pyrogenic exotoxins A, B,and C, relaxin, renin, SCF, soluble complement receptor 1, soluble I-CAM1, soluble interleukin receptors (IL-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12,13, 14, 15), soluble TNF receptor, somatomedin, somatostatin,somatotropin, streptokinase, superantigens, i.e., staphylococcalenterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE), superoxidedismutase, toxic shock syndrome toxin (TSST-1), thymosin alpha 1, tissueplasminogen activator, tumor necrosis factor (TNF β), tumor necrosisfactor receptor (TNFR), tumor necrosis factor-alpha (TNFα), vascularendothelial growth factor (VEGF), urokinase and others. These antigenscan be obtained by methods known to those of skill in the art, forexample, from commercial sources or from published polypeptide orpolynucleotide sequences (e.g. Genbank).

Additional antigens include, but are not limited to, transcriptional andexpression activators. Exemplary transcriptional and expressionactivators include genes and proteins that modulate cell growth,differentiation, regulation, or the like. Expression and transcriptionalactivators are found in prokaryotes, viruses, and eukaryotes, includingfungi, plants, and animals, including mammals, providing a wide range oftherapeutic targets. It will be appreciated that expression andtranscriptional activators regulate transcription by many mechanisms,e.g., by binding to receptors, stimulating a signal transductioncascade, regulating expression of transcription factors, binding topromoters and enhancers, binding to proteins that bind to promoters andenhancers, unwinding DNA, splicing pre-mRNA, polyadenylating RNA, anddegrading RNA. Antigens include, but are not limited to, expressionactivators such as cytokines, inflammatory molecules, growth factors,their receptors, and oncogene products, e.g., interleukins (e.g., IL-1,IL-2, IL-8, etc.), interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF,TGF-α, TGF-β, EGF, KGF, SCF/c-Kit, CD40L/CD40, VLA-4VCAM-1,ICAM-1/LFA-1, and hyalurin/CD44; signal transduction molecules andcorresponding oncogene products, e.g., Mos, Ras, Raf, and Met; andtranscriptional activators and suppressors, e.g., p53, Tat, Fos, Myc,Jun, Myb, Rel, and steroid hormone receptors such as those for estrogen,progesterone, testosterone, aldosterone, the LDL receptor ligand andcorticosterone.

Antigens may also include, but are not limited to, proteins frominfectious fungi, e.g., Aspergillus, Candida species; bacteria,particularly E. coli, which serves a model for pathogenic bacteria, aswell as medically important bacteria such as Staphylococci (e.g.,aureus), or Streptococci (e.g., pneumoniae); protozoa such as sporozoa(e.g., Plasmodia), rhizopods (e.g., Entamoeba) and flagellates(Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viruses such as(+) RNA viruses (examples include Poxviruses e.g., vaccinia;picornaviruses, e.g. polio; Togaviruses, e.g., rubella; flaviviruses,e.g., HCV; and coronaviruses), (−) RNA viruses (e.g., Rhabdoviruses,e.g., VSV; Paramyxovimses, e.g., RSV; Orthomyxovimses, e.g., influenza;Bunyaviruses; and Arenaviruses), dsDNA viruses (Reoviruses, forexample), RNA to DNA viruses, i.e., Retroviruses, e.g., HIV and HTLV,and certain DNA to RNA viruses such as hepatitis B.

Antigens may be enzymes including, but not limited to, amidases, aminoacid racemases, acylases, dehalogenases, dioxygenases, diarylpropaneperoxidases, epimerases, epoxide hydrolases, esterases, isomerases,kinases, glucose isomerases, glycosidases, glycosyl transferases,haloperoxidases, monooxygenases (e.g., p450s), lipases, ligninperoxidases, nitrile hydratases, nitrilases, proteases, phosphatases,subtilisins, transaminase, and nucleases.

Agriculturally related proteins such as insect resistance proteins(e.g., the Cry proteins), starch and lipid production enzymes, plant andinsect toxins, toxin-resistance proteins, mycotoxin detoxificationproteins, plant growth enzymes (e.g., ribulose 1,5-bisphosphatecarboxylase/oxygenase, “RUBISCO”), lipoxygenase (LOX), andphosphoenolpyruvate (PEP) carboxylase may also be antigens.

For example, the antigen may be a disease-associated molecule, such astumor surface antigen such as B-cell idiotypes, CD20 on malignant Bcells, CD33 on leukemic blasts, and HER2/neu on breast cancer.Alternatively, the antigen may be a growth factor receptor. Examples ofthe growth factors include, but are not limited to, epidermal growthfactors (EGFs), transferrin, insulin-like growth factor, transforminggrowth factors (TGFs), interleukin-1, and interleukin-2. For example, ahigh expression of EGF receptors has been found in a wide variety ofhuman epithelial primary tumors. TGF-α has been found to mediate anautocrine stimulation pathway in cancer cells. Several murine monoclonalantibodies have been demonstrated to be able to bind EGF receptors,block the binding of ligand to EGF receptors, and inhibit proliferationof a variety of human cancer cell lines in culture and in xenograftmodels. Mendelsohn and Baselga (1995) Antibodies to growth factors andreceptors, in Biologic Therapy of Cancer, 2nd Ed., J B Lippincott,Philadelphia, pp. 607-623. Thus, antibodies may be used to treat avariety of cancers.

The antigen may also be cell surface protein or receptor associated withcoronary artery disease such as platelet glycoprotein IIb/IIIa receptor,autoimmune diseases such as CD4, CAMPATH-1 and lipid A region of thegram-negative bacterial lipopolysaccharide. Humanized antibodies againstCD4 have been tested in clinical trials in the treatment of patientswith mycosis fungoides, generalized postular psoriasis, severepsoriasis, and rheumatoid arthritis. Antibodies against lipid A regionof the gram-negative bacterial lipopolysaccharide have been testedclinically in the treatment of septic shock. Antibodies againstCAMPATH-1 have also been tested clinically in the treatment of againstrefractory rheumatoid arthritis. Thus, antibodies provided herein may beused to treat a variety of autoimmune diseases.

Useful antigens also include proteins or peptides associated with humanallergic diseases, such as inflammatory mediator proteins, e.g.interleukin-1 (IL-1), tumor necrosis factor (TNF), leukotriene receptorand 5-lipoxygenase, and adhesion molecules such as V-CAM/VLA-4. Inaddition, IgE may also serve as the antigen because IgE plays pivotalrole in type I immediate hypersensitive allergic reactions such asasthma. Studies have shown that the level of total serum IgE tends tocorrelate with severity of diseases, especially in asthma. Burrows etal. (1989) “Association of asthma with serum IgE levels and skin-testreactivity to allergens” New Engl. L. Med. 320:271-277. Thus, antibodiesselected against IgE may be used to reduce the level of IgE or block thebinding of IgE to mast cells and basophils in the treatment of allergicdiseases without having substantial impact on normal immune functions.

The antigen may also be a viral surface or core protein which may serveas an antigen to trigger an immune response of the infected host.Examples of these viral proteins include, but are not limited to,glycoproteins (or surface antigens, e.g., GP120 and GP41) and capsidproteins (or structural proteins, e.g., P24 protein); surface antigensor core proteins of hepatitis A, B, C, D or E virus (e.g. smallhepatitis B surface antigen (SHBsAg) of hepatitis B virus and the coreproteins of hepatitis C virus, NS3, NS4 and NS5 antigens); glycoprotein(G-protein) or the fusion protein (F-protein) of respiratory syncytialvirus (RSV); surface and core proteins of herpes simplex virus HSV-1 andHSV-2 (e.g., glycoprotein D from HSV-2).

The antigen may also be a mutated tumor suppressor gene product that haslost its tumor-suppressing function and may render the cells moresusceptible to cancer. Tumor suppressor genes are genes that function toinhibit the cell growth and division cycles, thus preventing thedevelopment of neoplasia. Mutations in tumor suppressor genes cause thecell to ignore one or more of the components of the network ofinhibitory signals, overcoming the cell cycle check points and resultingin a higher rate of controlled cell growth-cancer. Examples of the tumorsuppressor genes include, but are not limited to, DPC-4, NF-1, NF-2, RB,p53, WT1, BRCA1 and BRCA2. DPC-4 is involved in pancreatic cancer andparticipates in a cytoplasmic pathway that inhibits cell division. NF-1codes for a protein that inhibits Ras, a cytoplasmic inhibitory protein.NF-1 is involved in neurofibroma and pheochromocytomas of the nervoussystem and myeloid leukemia. NF-2 encodes a nuclear protein that isinvolved in meningioma, schwanoma, and ependymoma of the nervous system.RB codes for the pRB protein, a nuclear protein that is a majorinhibitor of cell cycle. RB is involved in retinoblastoma as well asbone, bladder, small cell lung and breast cancer. p53 codes for p53protein that regulates cell division and can induce apoptosis. Mutationand/or inaction of p53 is found in a wide ranges of cancers. WT1 isinvolved in Wilms tumor of the kidneys. BRCA1 is involved in breast andovarian cancer, and BRCA2 is involved in breast cancer. Thus, antibodiesmay be used to block the interactions of the gene product with otherproteins or biochemicals in the pathways of tumor onset and development.

The antigen may be a CD molecule including but not limited to, CD1a,CD1b, CD1c, CD1d, CD2, CD3γ, CD3δ, CD3ε, CD4, CD5, CD6, CD7, CD8α, CD8β,CD9, CD10, CD11a, CD11b, CD11c, CDw12, CD13, CD14, CD15, CD15s, CD16a,CD16b, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28,CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40,CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45R, CD46, CD47,CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53,CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L, CD62P,CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68,CD69, CDw70, CD71, CD72, CD73, CD74, CDw75, CDw76, CD77, CD79a, CD79r3,CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91,CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102,CD103, CD104, CD105, CD106, CD107a, CD107b, CDw108, CDw109, CD110-113,CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a,CD121b, CD122, CD123, CDw124, CD125, CD126, CDw127, CDw128a, CDw128b,CD129, CDw130, CD131, CD132, CD133, CD134, CD135, CD136, CDw137, CD138,CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CDw145, CD146, CD147,CD148, CDw149, CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD157,CD158a, CD158b, CD161, CD162, CD163, CD164, CD165, CD166, and TCR4. Theantigen may be VEGF, VEGF receptor, EGFR, Her2, TNFa, TNFRI receptor,GPIIb/IIIa, IL-2Rα chain, IL-2R β chain, RSV F protein, α4 integrin,IgE, IgE receptor, digoxin, carpet viper venom, complement C5, OPGL,CA-125 tumor antigen, Staphylococci proteins, Staphylococcus epidermidisproteins, Staphylococcus aureus proteins, proteins involvedStaphylococcal infection (including but not limited to, Staphylococcusaureus and Staphylococcus epidermidis), IL-6 receptor, CTLA-4, RSV, Tacsubunit of IL-2 receptor, IL-5, and EpCam. The antigen may be a fragmentof a molecule.

In some embodiments, the parent antibodies are multispecific antibodies.Any suitable multispecific antibody may be used, including a bispecificantibody. Examples of useful bispecific parent antibodies include, butare not limited to, those with one antibody directed against a tumorcell antigen and the other antibody directed against a cytotoxic triggermolecule such as anti-FcγRI/anti-CD 15, anti-p185^(HER2)/FcγRIII (CD16),anti-CD3/anti-malignant B-cell (1D10), anti-CD3/anti-p185^(HER2),anti-CD3/anti-p97, anti-CD3/anti-renal cell carcinoma,anti-CD3/anti-OVCAR-3, anti-CD3/L-D1 (anti-colon carcinoma),anti-CD3/anti-melanocyte stimulating hormone analog, anti-EGFreceptor/anti-CD3, anti-CD3/anti-CAMA1, anti-CD3/anti-CD19,anti-CD3/MoV18, anti-neural cell adhesion molecule (NCAM)/anti-CD3,anti-folate binding protein (FBP)/anti-CD3, anti-pan carcinomaassociated antigen (AMOC-31)/anti-CD3; bispecific antibodies with oneantibody which binds specifically to a tumor antigen and anotherantibody which binds to a toxin such as anti-saporin/anti-Id-1,anti-CD22/anti-saporin, anti-CD7/anti-saporin, anti-CD38/anti-saporin,anti-CEA/anti-ricin A chain, anti-interferon-α (IFN-α)/anti-hybridomaidiotype, anti-CEA/anti-vinca alkaloid; bispecific antibodies forconverting enzyme activated prodrugs such as anti-CD30/anti-alkalinephosphatase (which catalyzes conversion of mitomycin phosphate prodrugto mitomycin alcohol); bispecific antibodies which can be used asfibrinolytic agents such as anti-fibrin/anti-tissue plasminogenactivator (tPA), anti-fibrin/anti-urokinase-type plasminogen activator(uPA); bispecific antibodies for targeting immune complexes to cellsurface receptors such as anti-low density lipoprotein (LDL)/anti-Fcreceptor (e.g. FcγRI, FcγRII or FcγRIII); bispecific antibodies for usein therapy of infectious diseases such as anti-CD3/anti-herpes simplexvirus (HSV), anti-T-cell receptor:CD3 complex/anti-influenza,anti-FcγR/anti-HIV; bispecific antibodies for tumor detection in vitroor in vivo such as anti-CEA/anti-EOTUBE, anti-CEA/anti-DPTA,anti-anti-p185^(HER2)/anti-hapten; bispecific antibodies as vaccineadjuvants (see Fanger, M W et al., Crit Rev Immunol. 1992;12(34):101-24, which is incorporated by reference herein); andbispecific antibodies as diagnostic tools such as anti-rabbitIgG/anti-ferritin, anti-horse radish peroxidase (HRP)/anti-hormone,anti-somatostatin/anti-substance P, anti-HRP/anti-FITC,anti-CEA/anti-β-galactosidase (see Nolan, O et R. O'Kennedy, BiochimBiophys Acta. 1990 Aug. 1; 1040(1):1-11, which is incorporated byreference herein). Examples of trispecific antibodies includeanti-CD3/anti-CD4/anti-CD37, anti-CD3/anti-CD5/anti-CD37 andanti-CD3/anti-CD8/anti-CD37.

Linkers and Payloads

One of skill in the art can use the reactive groups described herein tolink the polypeptides or antibodies to any molecular entity capable offorming a covalent bond with a modified amino acid described herein, ora modified amino acid residue derived from such a modified amino acid.Such linking can be performed directly or indirectly via a linker. Thus,provided herein are conjugates comprising a polypeptide comprising anamino acid residue corresponding to a compound of formula I, Ia, II,III, IV, V, VI, VII, VIII, IX, or 1-10 linked to a payload andoptionally comprising a linking moiety between the polypeptide and thepayload.

Useful linkers include those described herein. In certain embodiments,the linker is any divalent or multivalent linker known to those of skillin the art. Generally, the linker is capable of forming covalent bondswith the payload moiety and a modified amino acid or a modified aminoacid residue. Useful divalent linkers include a bond, alkylene,substituted alkylene, heteroalkylene, substituted heteroalkylene,arylene, substituted arylene, heteroarlyene and substitutedheteroarylene. In certain embodiments, the linker is C₁₋₁₀ alkylene orC₁₋₁₀ heteroalkylene.

The payload can be any molecular entity that one of skill in the artmight desire to conjugate to the polypeptide. In certain embodiments,the payload is a therapeutic moiety. In such embodiment, the polypeptideconjugate can be used to target the therapeutic moiety to its moleculartarget. In certain embodiments, the payload is a labeling moiety. Insuch embodiments, the polypeptide conjugate can be used to detectbinding of the polypeptide to its target. In certain embodiments, thepayload is a cytotoxic moiety. In such embodiments, the conjugate can beused target the cytotoxic moiety to a diseased cell, for example acancer cell, to initiate destruction or elimination of the cell.Conjugates comprising other payloads apparent to those of skill in theart are within the scope of the conjugates described herein.

In certain embodiments, a conjugate can have a payload selected from thegroup consisting of a label, a dye, a polymer, a water-soluble polymer,polyethylene glycol, a derivative of polyethylene glycol, aphotocrosslinker, a cytotoxic compound, a radionuclide, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a peptide, a water-soluble dendrimer, a cyclodextrin, aninhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, a photoisomerizablemoiety, biotin, a derivative of biotin, a biotin analogue, a moietyincorporating a heavy atom, a chemically cleavable group, aphotocleavable group, an elongated side chain, a carbon-linked sugar, aredox-active agent, an amino thioacid, a toxic moiety, an isotopicallylabeled moiety, a biophysical probe, a phosphorescent group, achemiluminescent group, an electron dense group, a magnetic group, anintercalating group, a chromophore, an energy transfer agent, abiologically active agent, a detectable label, a small molecule, or anycombination thereof. In an embodiment, the payload is a label, a dye, apolymer, a cytotoxic compound, a radionuclide, a drug, an affinitylabel, a resin, a protein, a polypeptide, a polypeptide analog, anantibody, antibody fragment, a metal chelator, a cofactor, a fatty acid,a carbohydrate, a polynucleotide, a DNA, a RNA, a peptide, afluorophore, or a carbon-linked sugar. In certain embodiments, thepayload is a label, a dye, a polymer, a drug, an antibody, antibodyfragment, a DNA, a RNA, or a peptide.

Useful drug payloads include any cytotoxic, cytostatic orimmunomodulatory agent. Useful classes of cytotoxic or immunomodulatoryagents include, for example, antitubulin agents, auristatins, DNA minorgroove binders, DNA replication inhibitors, alkylating agents (e.g.,platinum complexes such as cis-platin, mono(platinum), bis(platinum) andtri-nuclear platinum complexes and carboplatin), anthracyclines,antibiotics, antifolates, antimetabolites, calmodulin inhibitors,chemotherapy sensitizers, duocarmycins, etoposides, fluorinatedpyrimidines, ionophores, lexitropsins, maytansinoids, nitrosoureas,platinols, pore-forming compounds, purine antimetabolites, puromycins,radiation sensitizers, rapamycins, steroids, taxanes, topoisomeraseinhibitors, vinca alkaloids, or the like.

Individual cytotoxic or immunomodulatory agents include, for example, anandrogen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine,bleomycin, busulfan, buthionine sulfoximine, calicheamicin,calicheamicin derivatives, camptothecin, carboplatin, carmustine (BSNU),CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide,cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine,dactinomycin (formerly actinomycin), daunorubicin, decarbazine, DM1,DM4, docetaxel, doxorubicin, etoposide, an estrogen,5-fluordeoxyuridine, 5-fluorouracil, gemcitabine, gramicidin D,hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU),maytansine, mechlorethamine, melphalan, 6-mercaptopurine, methotrexate,mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel,palytoxin, plicamycin, procarbizine, rhizoxin, streptozotocin,tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine,vincristine, vinorelbine, VP-16 and VM-26.

In some embodiments, suitable cytotoxic agents include, for example, DNAminor groove binders (e.g., enediynes and lexitropsins, a CBI compound;see also U.S. Pat. No. 6,130,237), duocarmycins, taxanes (e.g.,paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38,topotecan, morpholino-doxorubicin, rhizoxin,cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin,epothilone A and B, estramustine, cryptophycins, cemadotin,maytansinoids, discodermolide, eleutherobin, and mitoxantrone.

In some embodiments, the payload is an anti-tubulin agent. Examples ofanti-tubulin agents include, but are not limited to, taxanes (e.g.,Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik) and vincaalkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine).Other antitubulin agents include, for example, baccatin derivatives,taxane analogs, epothilones (e.g., epothilone A and B), nocodazole,colchicine and colcimid, estramustine, cryptophycins, cemadotin,maytansinoids, combretastatins, discodermolide, and eleutherobin.

In certain embodiments, the cytotoxic agent is a maytansinoid, anothergroup of anti-tubulin agents. For example, in specific embodiments, themaytansinoid can be maytansine or DM-1 (ImmunoGen, Inc.; see also Chariet al., 1992, Cancer Res. 52:127-131).

In some embodiments, the payload is an auristatin, such as auristatin Eor a derivative thereof. For example, the auristatin E derivative can bean ester formed between auristatin E and a keto acid. For example,auristatin E can be reacted with paraacetyl benzoic acid orbenzoylvaleric acid to produce AEB and AEVB, respectively. Other typicalauristatin derivatives include AFP, MMAF, and MMAE. The synthesis andstructure of auristatin derivatives are described in U.S. PatentApplication Publication Nos. 2003-0083263, 2005-0238649 and2005-0009751; International Patent Publication No. WO 04/010957,International Patent Publication No. WO 02/088172, and U.S. Pat. Nos.6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149;5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191;5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278;4,816,444; and 4,486,414.

In some embodiments, the payload is not a radioisotope. In someembodiments, the payload is not radioactive.

In some embodiments, the payload is an antimetabolite. Theantimetabolite can be, for example, a purine antagonist (e.g.,azothioprine or mycophenolate mofetil), a dihydrofolate reductaseinhibitor (e.g., methotrexate), acyclovir, gangcyclovir, zidovudine,vidarabine, ribavarin, azidothymidine, cytidine arabinoside, amantadine,dideoxyuridine, iododeoxyuridine, poscarnet, or trifluridine.

In other embodiments, the payload is tacrolimus, cyclosporine, FU506 orrapamycin. In further embodiments, the drug is aldesleukin, alemtuzumab,alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenictrioxide, bexarotene, bexarotene, calusterone, capecitabine, celecoxib,cladribine, darbepoetin alfa, denileukin diftitox, dexrazoxanc,dromostanolone propionate, epirubicin, epoetin alfa, estramustine,exemestane, filgrastim, floxuridine, fludarabine, fulvestrant,gemcitabine, gemtuzumab ozogamicin (MYLOTARG), goserelin, idarubicin,ifosfamide, imatinib mesylate, interferon alfa-2a, irinotecan,letrozole, leucovorin, levamisole, meclorethamine or nitrogen mustard,megestrol, mesna, methotrexate, methoxsalen, mitomycin C, mitotane,nandrolone phenpropionate, oprelvekin, oxaliplatin, pamidronate,pegademase, pegaspargase, pegfilgrastim, pentostatin, pipobroman,plicamycin, porfimer sodium, procarbazine, quinacrine, rasburicase,Rituximab, sargramostim, streptozocin, tamoxifen, temozolomide,teniposide, testolactone, thioguanine, toremifene, tositumomab,trastuzumab (HERCEPTIN), tretinoin, uracil mustard, valrubicin,vinblastine, vincristine, vinorelbine or zoledronate.

In some embodiments, the payload is an immunomodulatory agent. Theimmunomodulatory agent can be, for example, gangcyclovir, etanercept,tacrolimus, cyclosporine, rapamycin, cyclophosphamide, azathioprine,mycophenolate mofetil or methotrexate. Alternatively, theimmunomodulatory agent can be, for example, a glucocorticoid (e.g.,cortisol or aldosterone) or a glucocorticoid analogue (e.g., prednisoneor dexamethasone).

In some embodiments, the immunomodulatory agent is an anti-inflammatoryagent, such as arylcarboxylic derivatives, pyrazole-containingderivatives, oxicam derivatives and nicotinic acid derivatives. Classesof anti-inflammatory agents include, for example, cyclooxygenaseinhibitors, 5-lipoxygenase inhibitors, and leukotriene receptorantagonists.

Suitable cyclooxygenase inhibitors include meclofenamic acid, mefenamicacid, carprofen, diclofenac, diflunisal, fenbufen, fenoprofen,indomethacin, ketoprofen, nabumetone, sulindac, tenoxicam and tolmetin.

Suitable lipoxygenase inhibitors include redox inhibitors (e.g.,catechol butane derivatives, nordihydroguaiaretic acid (NDGA),masoprocol, phenidone, Ianopalen, indazolinones, naphazatrom,benzofuranol, alkylhydroxylamine), and non-redox inhibitors (e.g.,hydroxythiazoles, methoxyalkylthiazoles, benzopyrans and derivativesthereof, methoxytetrahydropyran, boswellic acids and acetylatedderivatives of boswellic acids, and quinolinemethoxyphenylacetic acidssubstituted with cycloalkyl radicals), and precursors of redoxinhibitors.

Other suitable lipoxygenase inhibitors include antioxidants (e.g.,phenols, propyl gallate, flavonoids and/or naturally occurringsubstrates containing flavonoids, hydroxylated derivatives of theflavones, flavonol, dihydroquercetin, luteolin, galangin, orobol,derivatives of chalcone, 4,2′,4′-trihydroxychalcone, ortho-aminophenols,N-hydroxyureas, benzofuranols, ebselen and species that increase theactivity of the reducing selenoenzymes), iron chelating agents (e.g.,hydroxamic acids and derivatives thereof, N-hydroxyureas,2-benzyl-1-naphthol, catechols, hydroxylamines, carnosol trolox C,catechol, naphthol, sulfasalazine, zyleuton, 5-hydroxyanthranilic acidand 4-(omega-arylalkyl)phenylalkanoic acids), imidazole-containingcompounds (e.g., ketoconazole and itraconazole), phenothiazines, andbenzopyran derivatives.

Yet other suitable lipoxygenase inhibitors include inhibitors ofeicosanoids (e.g., octadecatetraenoic, eicosatetraenoic,docosapentaenoic, eicosahexaenoic and docosahexaenoic acids and estersthereof, PGE1 (prostaglandin E1), PGA2 (prostaglandin A2), viprostol,15-mo nohydroxyeicosatetraenoic, 15-monohydroxy-eicosatrienoic and15-monohydroxyeicosapentaenoic acids, and leukotrienes B5, C5 and D5),compounds interfering with calcium flows, phenothiazines,diphenylbutylamines, verapamil, fuscoside, curcumin, chlorogenic acid,caffeic acid, 5,8,11,14-eicosatetrayenoic acid (ETYA),hydroxyphenylretinamide, ionapalen, esculin, diethylcarbamazine,phenantroline, baicalein, proxicromil, thioethers, diallyl sulfide anddi-(1-propenyl) sulfide.

Leukotriene receptor antagonists include calcitriol, ontazolast, BayerBay-x-1005, Ciba-Geigy CGS-25019C, ebselen, Leo Denmark ETH-615, LillyLY-293111, Ono ONO-4057, Terumo TMK-688, Boehringer Ingleheim BI-RM-270,Lilly LY 213024, Lilly LY 264086, Lilly LY 292728, Ono ONO LB457, Pfizer105696, Perdue Frederick PF 10042, Rhone-Poulenc Rorer RP 66153,SmithKline Beecham SB-201146, SmithKline Beecham SB-201993, SmithKlineBeecham SB-209247, Searle SC-53228, Sumitamo SM 15178, American HomeProducts WAY 121006, Bayer Bay-o-8276, Warner-Lambert CI-987,Warner-Lambert CI-987BPC-15LY 223982, Lilly LY 233569, Lilly LY-255283,MacroNex MNX-160, Merck and Co. MK-591, Merck and Co. MK-886, OnoONO-LB-448, Purdue Frederick PF-5901, Rhone-Poulenc Rorer RG14893,Rhone-Poulenc Rorer RP 66364, Rhone-Poulenc Rorer RP 69698, ShionoogiS-2474, Searle SC-41930, Searle SC-50505, Searle SC-51146, SearleSC-52798, SmithKline Beecham SK&F-104493, Leo Denmark SR-2566, TanabeT-757 and Teijin TEI-1338.

Other useful drug payloads include chemical compounds useful in thetreatment of cancer. Examples of chemotherapeutic agents includeErlotinib (TARCEVA®. Genentech/OS1 Pharm.), Bortezomib (VELCADE®,Millennium Pharm.), Fulvestrant (FASLODEX®, AstraZeneca), Sutent(SU11248, Pfizer), Letrozole (FEMARA®, Novartis), Imatinib mesylate(GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin(Eloxatin®, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin(Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TYKERB®, GSK572016, GlaxoSmith Kline), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, BayerLabs), and Gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271;Sugen), alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlomaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin γ11 and calicheamicin omegall(Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin, includingdynemicin A; bisphosphonates, such as clodronate; an esperamicin; aswell as neocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin),morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamniprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®(paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® (doxetaxel; Rhone-Poulenc Rorer, Antony, France);chloranmbucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin;vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® (vinorelbine); novantrone; teniposide; edatrexate;daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; and pharmaceutically acceptable salts,acids and derivatives of any of the above.

Other useful payloads include: (i) anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene,droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,onapristone, and FARESTON® (toremifine citrate); (ii) aromataseinhibitors that inhibit the enzyme aromatase, which regulates estrogenproduction in the adrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane;Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA®(letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii)anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxo lane nucleosidecytosine analog); (iv) protein kinase inhibitors; (v) lipid kinaseinhibitors; (vi) antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in aberrantcell proliferation, such as, for example, PKC-α, Ralf and H-Ras; (vii)ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2expression inhibitors; (viii) vaccines such as gene therapy vaccines,for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; atopoisomerase 1 inhibitor such as LURTOTECAN®; ABARELIX® rmRH; (ix)anti-angiogenic agents such as bevacizumab (AVASTIN®, Genentech); and(x) pharmaceutically acceptable salts, acids and derivatives of any ofthe above. Other anti-angiogenic agents include MMP-2(matrix-metalloproteinase 2) inhibitors, MMP-9 (matrix-metalloproteinase9) inhibitors, COX-II (cyclooxygenase II) inhibitors, and VEGF receptortyrosine kinase inhibitors. Examples of such useful matrixmetalloproteinase inhibitors that can be used in combination with thepresent compounds/compositions are described in WO 96/33172, WO96/27583, EP 818442, EP 1004578, WO 98/07697, WO 98/03516, WO 98/34918,WO 98/34915, WO 98/33768, WO 98/30566, EP 606,046, EP 931,788, WO90/05719, WO 99/52910, WO 99/52889, WO 99/29667, WO 99/07675, EP 945864,U.S. Pat. No. 5,863,949, U.S. Pat. No. 5,861,510, and EP 780,386, all ofwhich are incorporated herein in their entireties by reference. Examplesof VEGF receptor tyrosine kinase inhibitors include4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474; Example 2 within WO 01/32651),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787; WO98/35985) and SU11248 (sunitinib; WO 01/60814), and compounds such asthose disclosed in PCT Publication Nos. WO 97/22596, WO 97/30035, WO97/32856, and WO 98/13354).

In certain embodiments, the payload is an antibody or an antibodyfragment. In certain embodiments, the payload antibody or fragment canbe encoded by any of the immunoglobulin genes recognized by those ofskill in the art. As described in more detail elsewhere in thisdisclosure, the immunoglobulin genes include, but are not limited to,the κ, λ, α, γ (IgG1, IgG2, IgG3, and IgG4), δ, ε and μ constant regiongenes, as well as the immunoglobulin variable region genes. The termincludes full-length antibody and antibody fragments recognized by thoseof skill in the art, and variants thereof. Exemplary fragments includebut are not limited to Fv, Fc, Fab, and (Fab′)₂, single chain Fv (scFv),diabodies, triabodies, tetrabodies, bifunctional hybrid polypeptides,CDR1, CDR2, CDR3, combinations of CDR's, variable regions, frameworkregions, constant regions, and the like.

In certain embodiments, the payload is one or more water-solublepolymers. A wide variety of macromolecular polymers and other moleculescan be linked to the polypeptides described herein to modulatebiological properties of the polypeptide, and/or provide new biologicalproperties to the polypeptide. These macromolecular polymers can belinked to the polypeptide via a naturally encoded amino acid or residue,via a modified amino acid or residue, or any functional substituent of anatural or modified amino acid or residue of either, or any substituentor functional group added to a natural or modified amino acid orresidue. The molecular weight of the polymer may be of a wide range,including but not limited to, between about 100 Da and about 100,000 Daor more.

The polymer selected may be water soluble so that a protein to which itis attached is more soluble or stable, or both, in an aqueousenvironment, such as a physiological environment. The polymer may bebranched or unbranched. Preferably, for therapeutic use of theend-product preparation, the polymer will be pharmaceuticallyacceptable.

The water soluble polymer may be any structural form including but notlimited to linear, forked or branched. Typically, the water solublepolymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG),but other water soluble polymers can also be employed. By way ofexample, PEG is used to describe certain embodiments.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Theterm “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented as linked to a polypeptide by the formula:XO—(CH₂CH₂O)_(n)—CH₂CH₂—Y where n is 2 to 10,000 and X is H or aterminal modification, including but not limited to, a C₁₋₄ alkyl, and Yis a group capable of forming a bond with the polypeptide.

In some cases, a PEG terminates on one end with hydroxy or methoxy,i.e., X is H or CH₃ (“methoxy PEG”). Alternatively, the PEG canterminate with a reactive group, thereby forming a bifunctional polymer.Typical reactive groups can include those reactive groups that arecommonly used to react with the functional groups found in the 20 commonamino acids (including but not limited to, maleimide groups, activatedcarbonates (including but not limited to, p-nitrophenyl ester),activated esters (including but not limited to, N-hydroxysuccinimide,p-nitrophenyl ester) and aldehydes) as well as functional groups thatare inert to the 20 common amino acids but that react specifically withcomplementary functional groups present in modified amino acids(including, but not limited to, tetrazine groups, strained alkenegroups, azide groups, and alkyne groups).

The other end of the PEG, which is shown in the above formula by Y, willattach either directly or indirectly to a polypeptide via anaturally-occurring or modified amino acid residue. For instance, Y maybe an amide, carbamate or urea capable of forming a linkage to an aminegroup (including but not limited to, the epsilon amine of lysine or theN-terminus) of the polypeptide. Alternatively, Y may be a maleimidecapable of forming a linkage to a thiol group (including but not limitedto, the thiol group of cysteine). In particular embodiments, Y may be astrained alkene capable of forming a linkage to a tetrazine group on amodified amino acid such as those described herein. In furtherembodiments, Y may be a tetrazine group capable of forming a linkage toa strained alkene group present in a modified amino acid residue. Y mayalso be an alkyne group capable of forming a linkage to an azide groupof a modified amino acid residue, or an azide group capable of forming alinkage to an alkyne group of a modified amino acid residue.

PEG of any suitable molecular mass may be used, including but notlimited to, from about 100 Daltons (Da) to 100,000 Da or more as desired(including but not limited to, sometimes 0.1-50 kDa or 10-40 kDa).Branched chain PEGs, including but not limited to, PEG molecules witheach chain having a MW ranging from 1-100 kDa (including but not limitedto, 1-50 kDa or 5-20 kDa) can also be used. A wide range of PEGmolecules are described in, including but not limited to, the ShearwaterPolymers, Inc. catalog, Nektar Therapeutics catalog, incorporated hereinby reference.

If the modified amino acid or residue comprises a tetrazine, then thePEG will typically contain a strained alkene. Alternatively, if themodified amino acid or residue comprises a strained alkene, such astrans-cyclooctene or norbornene, then the PEG will typically contain atetrazine. In preferred embodiments, the modified amino acid or residuecomprises the tetrazine and the PEG comprises the strained alkene. Inpreferred embodiments, the tetrazine functionality is provided on amodified amino acid as described herein.

As described above, other modified amino acids or residues may also beused to enable specific conjugation of different payloads. For example,if at least one modified amino acid or residue comprises an azide, thena PEG will typically contain either an alkyne moiety to effect formationof the [3+2] cycloaddition product or an activated PEG species (i.e.,ester, carbonate) containing a phosphine group to effect formation ofthe amide linkage. Alternatively, if the modified amino acid or residuecomprises an alkyne, then a PEG will typically contain an azide moietyto effect formation of the [3+2] Huisgen cycloaddition product.

If the modified amino acid or residue comprises a carbonyl group, thePEG will typically comprise a potent nucleophile (including but notlimited to, a hydrazide, hydrazine, hydroxylamine, or semicarbazidefunctionality) in order to effect formation of corresponding hydrazone,oxime, and semicarbazone linkages, respectively.

In certain embodiments, the payload is a strained alkene-containingpolymer comprising a water soluble polymer backbone having an averagemolecular weight from about 800 Da to about 100,000 Da. The polymerbackbone of the water-soluble polymer can be poly(ethylene glycol).However, it should be understood that a wide variety of water solublepolymers including but not limited to poly(ethylene)glycol and otherrelated polymers, including poly(dextran) and poly(propylene glycol),are also suitable for use and that the use of the term PEG orpoly(ethyleneglycol) is intended to encompass and include all suchmolecules. The term PEG includes, but is not limited to, poly(ethyleneglycol) in any of its forms, including bifunctional PEG, multiarmed PEG,derivatized PEG, forked PEG, branched PEG, pendent PEG (i.e. PEG orrelated polymers having one or more functional groups pendent to thepolymer backbone), or PEG with degradable linkages therein.

In certain embodiments, the proportion of polyethylene glycol moleculesto polypeptide molecules will vary, as will their concentrations in thereaction mixture. In general, the optimum ratio (in terms of efficiencyof reaction in that there is minimal excess unreacted protein orpolymer) may be determined by the molecular weight of the polyethyleneglycol selected and on the number of available reactive groupsavailable. As relates to molecular weight, typically the higher themolecular weight of the polymer, the fewer number of polymer moleculeswhich may be attached to the protein. Similarly, branching of thepolymer should be taken into account when optimizing these parameters.Generally, the higher the molecular weight (or the more branches) thehigher the polymer:protein ratio.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, glycerol oligomers, pentaerythritoland sorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(-PEG-OH)_(m) in which R is derived froma core moiety, such as glycerol, glycerol oligomers, or pentaerythritol,and m represents the number of arms. Multi-armed PEG molecules, such asthose described in U.S. Pat. Nos. 5,932,462 5,643,575; 5,229,490;4,289,872; U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259,each of which is incorporated by reference herein in its entirety, canalso be used as the polymer backbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(-YCHZ₂)_(n), where Y is a linking group and Z is an activatedterminal group linked to CH by a chain of atoms of defined length.

Yet another branched form, the pendant PEG, has reactive groups, such ascarboxyl, along the PEG backbone rather than at the end of PEG chains.

In addition to these forms of PEG, the polymer can also be prepared withweak or degradable linkages in the backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. As shown herein, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:-PEG-CO₂—PEG-+H₂O→PEG-CO₂H+HO-PEG- It is understood by those skilled inthe art that the term poly(ethylene glycol) or PEG represents orincludes all the forms known in the art including but not limited tothose disclosed herein.

Many other polymers are also suitable for use. In some embodiments,polymer backbones that are water-soluble, with from 2 to about 300termini, are particularly. Examples of suitable polymers include, butare not limited to, other poly(alkylene glycols), such as poly(propyleneglycol) (“PPG”), copolymers thereof (including but not limited tocopolymers of ethylene glycol and propylene glycol), terpolymersthereof, mixtures thereof, and the like. Although the molecular weightof each chain of the polymer backbone can vary, it is typically in therange of from about 800 Da to about 100,000 Da, often from about 6,000Da to about 80,000 Da.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble backbones is by no means exhaustiveand is merely illustrative, and that all polymeric materials having thequalities described herein are contemplated as being suitable for use.

In some embodiments the polymer derivatives are “multi-functional”,meaning that the polymer backbone has at least two termini, and possiblyas many as about 300 termini, functionalized or activated with afunctional group. Multifunctional polymer derivatives include, but arenot limited to, linear polymers having two termini, each terminus beingbonded to a functional group which may be the same or different.

The term “protected” refers to the presence of a protecting group ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also beused.

Specific examples of terminal functional groups in the literatureinclude, but are not limited to, N-succinimidyl carbonate (see e.g.,U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmann et al.Makromol. Chem. 182:1379 (1981), Zaplipsky et al. Eur. Polym. J. 19:1177(1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem. 179:301(1978)), succinimidyl propionate and succinimidyl butanoate (see, e.g.,Olson et al. in Poly(ethylene glycol) Chemistry & BiologicalApplications, pp. 170-181, Harris & Zaplipsky Eds., ACS, Washington,D.C., 1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate(See, e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) andJoppich et al. Macrolol. Chem. 180:1381 (1979), succinimidyl ester (see,e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S.Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. JBiochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354(1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal.Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251(1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl.Biochem. Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem.Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym.Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No.5,252,714), maleimide (see, e.g., Goodson et al. Bio/Technology 8:343(1990), Romani et al. in Chemistry of Peptides and Proteins 2:29(1984)), and Kogan, Synthetic Comm. 22:2417 (1992)),orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem.4:314(1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581(1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). All of theabove references and patents are incorporated herein by reference.

In certain embodiments, polymer derivatives comprise a polymer backbonehaving the structure: X—CH₂CH₂O—(CH₂CH₂O)_(n)CH₂CH₂—ZZ, wherein: X is afunctional group as described herein, n is about 20 to about 4000, andZZ is a moiety comprising a strained alkene. In some embodiments, thepolymer derivatives comprise a polymer backbone having the structure:X—CH₂CH₂O—(CH₂CH₂O)_(m)CH₂CH₂—O—(CH₂)_(m)—W—ZZ wherein: W is analiphatic or aromatic linker moiety comprising between 1-10 carbonatoms, n is about 20 to about 4000, X is a functional group as describedherein, m is between 1 and 10, and ZZ is a moiety comprising a strainedalkene. Examples of suitable functional groups include, but are notlimited to, hydroxyl, protected hydroxyl, acetal, alkenyl, amine,aminooxy, protected amine, protected hydrazide, protected thiol,carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine,and vinylpyridine, and ketone.

In some embodiments, the polymer derivatives comprise a polymer backbonehaving the structure: X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O_(n)—(CH₂)_(n)—Y,wherein: Y is a moiety comprising a strained alkene; X is a functionalgroup as described herein; n is about 20 to about 4000; and m is between1 and 10. Specific examples of each of the heterobifunctional PEGpolymers are shown herein.

In some embodiments, a strained alkene-terminal PEG derivative will havethe following structure: RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—Y where Y is amoiety comprising a strained alkene, R is a simple alkyl (methyl, ethyl,propyl, etc.), m is 2-10 and n is 100-1,000 (i.e., average molecularweight is between 5-40 kDa).

In some embodiments, a strained alkene-terminal PEG derivative will havethe following structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—(CH₂)_(p)—Y, where Y is a moietycomprising a strained alkene, R is a simple alkyl (methyl, ethyl,propyl, etc.), m is 2-10, p is 2-10 and n is 100 1,000.

Strained alkene-containing PEG derivatives can be prepared by a varietyof methods known in the art and/or disclosed herein. In a method forpreparation of a strained alkene-containing polymer derivative, alinking agent bearing a strained alkene functionality is contacted witha payload moiety, wherein the linking agent bears a chemicalfunctionality that will react selectively with a chemical functionalityon the PEG polymer, to form a strained alkene-containing polymerderivative product wherein the strained alkene is separated from thepolymer backbone by a linking group. Useful PEGs comprising strainedalkenes can be obtained from commercial sources, e.g. Jena Biosciences,or prepared according to published techniques, e.g. Aimetti et al.,2009, Biomaterials 30:6048-6054.

An exemplary reaction scheme is shown here:X-PEG-M+N-linker-Y→PG-X-PEG-linker-Y wherein: Y is a moiety comprising astrained alkene, PEG is poly(ethylene glycol) and X is a capping groupsuch as alkoxy or a functional group as described herein; and M is afunctional group that is not reactive with the strained alkenefunctionality but that will react efficiently and selectively with the Nfunctional group. Examples of suitable functional groups include, butare not limited to, M being a carboxylic acid, carbonate or active esterif N is an amine; M being a ketone if N is a hydrazide or aminooxymoiety; M being a leaving group if N is a nucleophile.

In one method for the preparation of a strained alkene-containing PEGderivative, a water soluble polymer backbone having an average molecularweight from about 800 Da to about 100,000 Da, the polymer backbonehaving a first terminus bonded to a first functional group and a secondterminus bonded to a suitable nucleophilic group, is reacted with acompound that bears both a strained alkene functionality and a leavinggroup that is suitable for reaction with the nucleophilic group on thePEG. When the PEG polymer bearing the nucleophilic moiety and themolecule bearing the leaving group are combined, the leaving groupundergoes a nucleophilic displacement and is replaced by thenucleophilic moiety, affording the desired strained alkene-containingpolymer: X-PEG-Nu+L-A-C→X-PEG-Nu-A-Y, where Y is a moiety comprising astrained alkene.

As shown, in some embodiments, a preferred polymer backbone for use inthe reaction has the formula X-PEG-Nu, wherein PEG is poly(ethyleneglycol), Nu is a nucleophilic moiety and X is a functional group thatdoes not react with Nu, L or the strained alkene functionality.

Examples of Nu include, but are not limited to, amine, alkoxy, aryloxy,sulfhydryl, imino, carboxylate, hydrazide, aminoxy groups that wouldreact primarily via a SN2-type mechanism. Additional examples of Nugroups include those functional groups that would react primarily via annucleophilic addition reaction. Examples of L groups include chloride,bromide, iodide, mesylate, tresylate, and tosylate and other groupsexpected to undergo nucleophilic displacement as well as ketones,aldehydes, thioesters, olefins, alpha-beta unsaturated carbonyl groups,carbonates and other electrophilic groups expected to undergo additionby nucleophiles.

In certain embodiments, A is an aliphatic linker of between 1-10 carbonatoms or a substituted aryl ring of between 6-14 carbon atoms. X is afunctional group which does not react with strained alkene groups and Lis a suitable leaving group.

In another method for preparation of the strained alkene-containingpolymer derivatives, a PEG polymer having an average molecular weightfrom about 800 Da to about 100,000 Da, bearing either a protectedfunctional group or a capping agent at one terminus and a suitableleaving group at the other terminus is contacted by an activatedmolecule comprising a strained alkene.

Purification of the crude products may be accomplished by known methodsincluding, but are not limited to, precipitation of the product followedby chromatography, if necessary.

A more specific example is the case of PEG diamine, in which one of theamines is protected by a protecting group moiety such as tert-butyl-Bocand the resulting mono-protected PEG diamine is reacted with a linkingmoiety that bears the strained alkene functionality:BocHN-PEG-NH₂+HO₂C—(CH₂)₃—[ZZ], where ZZ is a moiety comprising astrained alkene. In this instance, the amine group can be coupled to thecarboxylic acid group using a variety of activating agents such asthionyl chloride or carbodiimide reagents and N-hydroxysuccinimide orN-hydroxybenzotriazole to create an amide bond between the monoamine PEGderivative and the strained alkene-bearing linker moiety. Aftersuccessful formation of the amide bond, the resultingN-tert-butyl-Boc-protected derivative can be used directly to modifybioactive molecules or it can be further elaborated to install otheruseful functional groups. For instance, the N-t-Boc group can behydrolyzed by treatment with strong acid to generate an omega-amino-PEGderivative. The resulting amine can be used as a synthetic handle toinstall other useful functionality such as maleimide groups, activateddisulfides, activated esters and so forth for the creation of valuableheterobifunctional reagents.

In certain embodiments, the polymer derivative has the structure:X-A-PAY—B—Y, where Y is a moiety comprising a strained alkene; B is alinking moiety, which may be present or absent; PAY is a payload moiety;A is a linking moiety, which may be present or absent and which may bethe same as B or different; and X is a second functional group.

Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and morepreferably between 1-10 carbon atoms. A heteroatom such as nitrogen,oxygen or sulfur may be included with the alkyl chain. The alkyl chainmay also be branched at a heteroatom. Other examples of a linking moietyfor A and B include, but are not limited to, a multiply functionalizedaryl group, containing up to 10 and more preferably 5-6 carbon atoms.The aryl group may be substituted with one more carbon atoms, nitrogen,oxygen, or sulfur atoms. Other examples of suitable linking groupsinclude those linking groups described in U.S. Pat. Nos. 5,932,462 and5,643,575 and U.S. Pat. Appl. Publication 2003/0143596, each of which isincorporated by reference herein. Those of ordinary skill in the artwill recognize that the foregoing list for linking moieties is by nomeans exhaustive and is intended to be merely illustrative, and that awide variety of linking moieties having the qualities described hereinare contemplated to be useful.

Examples of suitable functional groups for use as X include hydroxyl,protected hydroxyl, alkoxyl, active ester, such as N-hydroxysuccinimidylesters and 1-benzotriazolyl esters, active carbonate, such asN-hydroxysuccinimidyl carbonates and 1-benzotriazolyl carbonates,acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, ketone, and acetylene. Aswould be understood, the selected X moiety should be compatible with thestrained alkene group so that reaction with the strained alkene groupdoes not occur. The strained alkene-containing polymer derivatives maybe homobifunctional, meaning that the second functional group (i.e., X)is also an strained alkene moiety, or heterobifunctional, meaning thatthe second functional group is a different functional group.

Water soluble polymers can be linked to the polypeptides. The watersoluble polymers may be linked via a modified amino acid residueincorporated in the polypeptides or any functional group or substituentof a modified or naturally encoded amino acid residue, or any functionalgroup or substituent added to a modified or naturally encoded amino acidresidue. In an embodiment, the modified amino acid residue is a modifiedamino acid residue as described herein. Alternatively, the water solublepolymers are linked to an antigen-binding antibody incorporating amodified amino acid residue via a naturally-occurring amino acid residue(including but not limited to, cysteine, lysine or the amine group ofthe N-terminal residue). In some cases, the polypeptides comprise 1, 2,3, 4, 5, 6, 7, 8, 9, 10 modified amino acid residues, wherein one ormore modified amino acid residues are linked to water soluble polymer(s)(including but not limited to, PEG and/or oligosaccharides). In somecases, the polypeptides further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more naturally-encoded amino acid residues linked to water solublepolymers. In some cases, the polypeptides comprise one or more modifiedamino acid residues linked to water soluble polymers and one or morenaturally-occurring amino acid residues linked to water solublepolymers. In some embodiments, the water soluble polymers enhance theserum half-life of the polypeptides relative to the unconjugated form.

The number of water soluble polymers linked to a polypeptide (i.e., theextent of PEGylation or glycosylation) can be adjusted to provide analtered (including but not limited to, increased or decreased)pharmacologic, pharmacokinetic or pharmacodynamic characteristic such asin vivo half-life. In some embodiments, the half-life of a polypeptideis increased at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 percent,2-fold, 5-fold, 10-fold, 50-fold, or at least about 100-fold over anunmodified polypeptide.

The degree and sites at which water soluble polymer(s) are linked to thepolypeptides can modulate the binding of the polypeptides to an antigenor receptor.

PEGylation (i.e., addition of any water soluble polymer) of polypeptidescontaining a modified amino acid residue, such as a tetrazine-comprisingamino acid residue, is carried out by any convenient method. Forexample, a polypeptide may be PEGylated with a strainedalkene-terminated PEG derivative. Briefly, an excess of solid PEG-Y,wherein Y is a moiety comprising a strained alkene, is added, withstirring, to an aqueous solution of a polypeptide comprising an aminoacid residue comprising a tetrazine functional group (such as a modifiedamino acid described herein), at room temperature. Typically, theaqueous solution is buffered with a buffer having a pK_(a) near the pHat which the reaction is to be carried out (generally about pH 4-10).Examples of suitable buffers for PEGylation at pH 7.5, for instance,include, but are not limited to, HEPES, phosphate, borate, TRIS-HCl,EPPS, and TES. The pH is continuously monitored and adjusted ifnecessary. The reaction is typically allowed to continue for betweenabout 1-48 hours.

The reaction products are subsequently subjected to hydrophobicinteraction chromatography to separate the PEGylated polypeptidevariants from free PEG-Y and any high-molecular weight complexes of thePEGylated polypeptide which may form when unblocked PEG is activated atboth ends of the molecule, thereby crosslinking polypeptide variantmolecules. The conditions during hydrophobic interaction chromatographyare such that free PEG-Y flows through the column, while any crosslinkedPEGylated polypeptide variant complexes elute after the desired forms,which contain one polypeptide variant molecule conjugated to one or morePEG groups. Suitable conditions vary depending on the relative sizes ofthe cross-linked complexes versus the desired conjugates and are readilydetermined by those skilled in the art. The eluent containing thedesired conjugates is concentrated by ultrafiltration and desalted bydiafiltration.

If necessary, the PEGylated polypeptide obtained from the hydrophobicchromatography can be purified further by one or more procedures knownto those skilled in the art including, but are not limited to, affinitychromatography; anion- or cation-exchange chromatography (using,including but not limited to, DEAE SEPHAROSE); chromatography on silica;reverse phase HPLC; gel filtration (using, including but not limited to,SEPHADEX G-75); hydrophobic interaction chromatography; size-exclusionchromatography, metal-chelate chromatography;ultrafiltration/diafiltration; ethanol precipitation; ammonium sulfateprecipitation; chromatofocusing; displacement chromatography;electrophoretic procedures (including but not limited to preparativeisoelectric focusing), differential solubility (including but notlimited to ammonium sulfate precipitation), or extraction. Apparentmolecular weight may be estimated by GPC by comparison to globularprotein standards (PROTEIN PURIFICATION METHODS, A PRACTICAL APPROACH(Harris & Angal, Eds.) IRL Press 1989, 293-306). The purity of thepolypeptide-PEG conjugate can be assessed by proteolytic degradation(including but not limited to, trypsin cleavage) followed by massspectrometry analysis. Pepinsky B., et al., J. Pharmcol. & Exp. Ther.297(3):1059-66 (2001).

A water soluble polymer linked to an amino acid residue of a polypeptidecan be further derivatized or substituted without limitation.

In certain embodiments, a polypeptide comprising a tetrazine-containingamino acid residue is modified with a branched PEG derivative thatcontains a terminal strained alkene moiety, with each chain of thebranched PEG having a MW ranging from 10-40 kDa and, more preferably,from 5-20 kDa. For instance, in some embodiments, the strainedalkene-terminal PEG derivative will have the following structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—(CH₂)_(p)Y where Y isa moiety comprising a strained alkene, R is a simple alkyl (methyl,ethyl, propyl, etc.), m is 2-10, p is 2-10, and n is 100-1,000, and X isoptionally an O, N, S or carbonyl group (C═O), or not present.

Useful exemplary PEG molecules that may be linked to polypeptides, aswell as PEGylation methods include those described in, e.g., U.S. PatentPublication Nos. 2004/0001838; 2002/0052009; 2003/0162949; 2004/0013637;2003/0228274; 2003/0220447; 2003/0158333; 2003/0143596; 2003/0114647;2003/0105275; 2003/0105224; 2003/0023023; 2002/0156047; 2002/0099133;2002/0086939; 2002/0082345; 2002/0072573; 2002/0052430; 2002/0040076;2002/0037949; 2002/0002250; 2001/0056171; 2001/0044526; 2001/0027217;2001/0021763; U.S. Pat. Nos. 6,646,110; 5,824,778; 5,476,653; 5,219,564;5,629,384; 5,736,625; 4,902,502; 5,281,698; 5,122,614; 5,473,034;5,516,673; 5,382,657; 6,552,167; 6,610,281; 6,515,100; 6,461,603;6,436,386; 6,214,966; 5,990,237; 5,900,461; 5,739,208; 5,672,662;5,446,090; 5,808,096; 5,612,460; 5,324,844; 5,252,714; 6,420,339;6,201,072; 6,451,346; 6,306,821; 5,559,213; 5,612,460; 5,747,646;5,834,594; 5,849,860; 5,980,948; 6,004,573; 6,129,912; WO 97/32607, EP229,108, EP 402,378, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039,WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131,WO 98/05363, EP 809 996, WO 96/41813, WO 96/07670, EP 605 963, EP 510356, EP 400 472, EP 183 503 and EP 154 316, which are incorporated byreference herein. Any of the PEG molecules described herein may be usedin any form, including but not limited to, single chain, branched chain,multiarm chain, single functional, bi-functional, multi-functional, orany combination thereof.

In certain embodiments, the polypeptides can be linked to the payloadswith one or more linkers capable of reacting with the modified aminoacid residue. The one or more linkers can be any linkers apparent tothose of skill in the art. The term “linker” is used herein to refer togroups or bonds that normally are formed as the result of a chemicalreaction and typically are covalent linkages. Hydrolytically stablelinkages means that the linkages are substantially stable in water anddo not react with water at useful pH values, including but not limitedto, under physiological conditions for an extended period of time,perhaps even indefinitely. Hydrolytically unstable or degradablelinkages mean that the linkages are degradable in water or in aqueoussolutions, including for example, blood. Enzymatically unstable ordegradable linkages mean that the linkage can be degraded by one or moreenzymes. As understood in the art, PEG and related polymers may includedegradable linkages in the polymer backbone or in the linker groupbetween the polymer backbone and one or more of the terminal functionalgroups of the polymer molecule. For example, ester linkages formed bythe reaction of PEG carboxylic acids or activated PEG carboxylic acidswith alcohol groups on a biologically active agent generally hydrolyzeunder physiological conditions to release the agent. Otherhydrolytically degradable linkages include, but are not limited to,carbonate linkages; imine linkages resulted from reaction of an amineand an aldehyde; phosphate ester linkages formed by reacting an alcoholwith a phosphate group; hydrazone linkages which are reaction product ofa hydrazide and an aldehyde; acetal linkages that are the reactionproduct of an aldehyde and an alcohol; orthoester linkages that are thereaction product of a formate and an alcohol; peptide linkages formed byan amine group, including but not limited to, at an end of a polymersuch as PEG, and a carboxyl group of a peptide; and oligonucleotidelinkages formed by a phosphoramidite group, including but not limitedto, at the end of a polymer, and a 5′ hydroxyl group of anoligonucleotide. Branched linkers may be used in polypeptides. A numberof different cleavable linkers are known to those of skill in the art.See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. The mechanismsfor release of an agent from these linker groups include, for example,irradiation of a photolabile bond and acid-catalyzed hydrolysis. U.S.Pat. No. 4,671,958, for example, includes a description ofimmunoconjugates comprising linkers which are cleaved at the target sitein vivo by the proteolytic enzymes of the patient's complement system.The length of the linker may be predetermined or selected depending upona desired spatial relationship between the polypeptide and the moleculelinked to it. In view of the large number of methods that have beenreported for attaching a variety of radiodiagnostic compounds,radiotherapeutic compounds, drugs, toxins, and other agents topolypeptides one skilled in the art will be able to determine a suitablemethod for attaching a given agent to a polypeptide

Any hetero- or homo-bifunctional linker can be used to link the payloadto the polypeptide or antibody. The linker may have a wide range ofmolecular weight or molecular length. Larger or smaller molecular weightlinkers may be used to provide a desired spatial relationship orconformation between the polypeptide and the linked entity. Linkershaving longer or shorter molecular length may also be used to provide adesired space or flexibility between the polypeptide and the linkedentity. Similarly, a linker having a particular shape or conformationmay be utilized to impart a particular shape or conformation to thepolypeptide or the linked entity, either before or after the polypeptidereaches its target. The functional groups present on each end of thelinker may be selected to modulate the release of a polypeptide or apayload under desired conditions. This optimization of the spatialrelationship between the polypeptide and the linked entity may providenew, modulated, or desired properties to the molecule.

In some embodiments, provided herein water-soluble bifunctional linkersthat have a dumbbell structure that includes: a) a tetrazine, a strainedalkene, or a carbonyl-containing moiety on at least a first end of apolymer backbone; and b) at least a second functional group on a secondend of the polymer backbone. The second functional group can be the sameor different as the first functional group. The second functional group,in some embodiments, is not reactive with the first functional group. Insome embodiments, water-soluble compounds that comprise at least one armof a branched molecular structure are provided. For example, thebranched molecular structure can be a dendritic structure.

Polypeptide Compositions

Polypeptides described herein can be formulated into compositions usingmethods available in the art and those disclosed herein. Any of thecompounds disclosed herein can be provided in the appropriatepharmaceutical composition and be administered by a suitable route ofadministration.

In certain embodiments, the polypeptide compositions provided hereinfurther comprise a pharmaceutically acceptable carrier. The carrier canbe a diluent, excipient, or vehicle with which the pharmaceuticalcomposition is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in E. W. Martin, 1990,Remington's Pharmaceutical Sciences, Mack Publishing Co.

In some embodiments, the pharmaceutical composition is provided in aform suitable for administration to a human subject. In someembodiments, the pharmaceutical composition will contain aprophylactically or therapeutically effective amount of the polypeptidetogether with a suitable amount of carrier so as to provide the form forproper administration to the patient. The formulation should suit themode of administration.

In some embodiments, the pharmaceutical composition is provided in aform suitable for intravenous administration. Typically, compositionssuitable for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Such compositions, however,may be administered by a route other than intravenous administration.

In particular embodiments, the pharmaceutical composition is suitablefor subcutaneous administration. In particular embodiments, thepharmaceutical composition is suitable for intramuscular administration.

Components of the pharmaceutical composition can be supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate. Where the composition isto be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ample of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

In some embodiments, the pharmaceutical composition is supplied as a drysterilized lyophilized powder that is capable of being reconstituted tothe appropriate concentration for administration to a subject. In someembodiments, polypeptides are supplied as a water free concentrate. Insome embodiments, the polypeptide is supplied as a dry sterilelyophilized powder at a unit dosage of at least 0.5 mg, at least 1 mg,at least 2 mg, at least 3 mg, at least 5 mg, at least 10 mg, at least 15mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 45 mg, atleast 50 mg, at least 60 mg, or at least 75 mg.

In certain embodiments, the pharmaceutical composition is supplied inliquid form. In some embodiments, the pharmaceutical composition isprovided in liquid form and is substantially free of surfactants and/orinorganic salts. In some embodiments, the polypeptide is supplied as inliquid form at a unit dosage of at least 0.1 mg/ml, at least 0.5 mg/ml,at least 1 mg/ml, at least 2.5 mg/ml, at least 3 mg/ml, at least 5mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least25 mg/ml, at least 30 mg/ml, or at least 60 mg/ml.

In some embodiments, the pharmaceutical composition is formulated as asalt form. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

In therapeutic use, the practitioner will determine the posology mostappropriate according to a preventive or curative treatment andaccording to the age, weight, stage of the infection and other factorsspecific to the subject to be treated. In certain embodiments, doses arefrom about 1 to about 1000 mg per day for an adult, or from about 5 toabout 250 mg per day or from about 10 to 50 mg per day for an adult. Incertain embodiments, doses are from about 5 to about 400 mg per day or25 to 200 mg per day per adult. In certain embodiments, dose rates offrom about 50 to about 500 mg per day are also contemplated.

Methods of Use for Therapy or Prophylaxis

Certain polypeptides provided herein can be used for the treatment orprevention of any disease or condition deemed suitable to thepractitioner of skill in the art. Generally, a method of treatment orprevention encompasses the administration of a therapeutically orprophylactically effective amount of the polypeptide or polypeptidecomposition to a subject in need thereof to treat or prevent the diseaseor condition.

A therapeutically effective amount of the polypeptide or composition isan amount that is effective to reduce the severity, the duration and/orthe symptoms of a particular disease or condition. The amount of thepolypeptide or composition that will be therapeutically effective in theprevention, management, treatment and/or amelioration of a particulardisease can be determined by standard clinical techniques. The preciseamount of the polypeptide or composition to be administered with depend,in part, on the route of administration, the seriousness of theparticular disease or condition, and should be decided according to thejudgment of the practitioner and each subject's circumstances.

In some embodiments, the effective amount of the polypeptide providedherein is between about 0.025 mg/kg and about 1000 mg/kg body weight ofa human subject. In certain embodiments, the polypeptide is administeredto a human subject at an amount of about 1000 mg/kg body weight or less,about 950 mg/kg body weight or less, about 900 mg/kg body weight orless, about 850 mg/kg body weight or less, about 800 mg/kg body weightor less, about 750 mg/kg body weight or less, about 700 mg/kg bodyweight or less, about 650 mg/kg body weight or less, about 600 mg/kgbody weight or less, about 550 mg/kg body weight or less, about 500mg/kg body weight or less, about 450 mg/kg body weight or less, about400 mg/kg body weight or less, about 350 mg/kg body weight or less,about 300 mg/kg body weight or less, about 250 mg/kg body weight orless, about 200 mg/kg body weight or less, about 150 mg/kg body weightor less, about 100 mg/kg body weight or less, about 95 mg/kg body weightor less, about 90 mg/kg body weight or less, about 85 mg/kg body weightor less, about 80 mg/kg body weight or less, about 75 mg/kg body weightor less, about 70 mg/kg body weight or less, or about 65 mg/kg bodyweight or less.

In some embodiments, the effective amount of polypeptide provided hereinis between about 0.025 mg/kg and about 60 mg/kg body weight of a humansubject. In some embodiments, the effective amount of a polypeptide ofthe pharmaceutical composition provided herein is about 0.025 mg/kg orless, about 0.05 mg/kg or less, about 0.10 mg/kg or less, about 0.20mg/kg or less, about 0.40 mg/kg or less, about 0.80 mg/kg or less, about1.0 mg/kg or less, about 1.5 mg/kg or less, about 3 mg/kg or less, about5 mg/kg or less, about 10 mg/kg or less, about 15 mg/kg or less, about20 mg/kg or less, about 25 mg/kg or less, about 30 mg/kg or less, about35 mg/kg or less, about 40 mg/kg or less, about 45 mg/kg or less, about50 mg/kg or about 60 mg/kg or less.

The pharmaceutical composition of the method can be administered usingany method known to those skilled in the art. For example, thepharmaceutical composition can be administered intramuscularly,intradermally, intraperitoneally, intravenously, subcutaneouslyadministration, or any combination thereof. In some embodiments, thepharmaceutical composition is administered subcutaneously. In someembodiments, the composition is administered intravenously. In someembodiments, the composition is administered intramuscularly.

Methods of Use for Detection or Diagnosis

The polypeptides provided herein can be used for the detection of anytarget or for the diagnosis of any disease or condition deemed suitableto the practitioner of skill in the art. The methods encompass detectingthe binding of a polypeptide to a target in an appropriate location,e.g., the appropriate body, tissue, or cell. In the methods, theformation of a complex between the polypeptide and target can bedetected by any method known to those of skill in the art. Examplesinclude assays that use secondary reagents for detection, ELISA's andimmunoprecipitation and agglutination assays. A detailed description ofthese assays is, for example, given in Harlow and Lane, Polypeptides: ALaboratory Manual (Cold Spring Harbor Laboratory, New York 1988 555-612,WO96/13590 to Maertens and Stuyver, Zrein et al. (1998) and WO96/29605.

For in situ diagnosis, the polypeptide may be administered to a subjectby methods known in the art such as, for example, intravenous,intranasal, intraperitoneal, intracerebral, intraarterial injection suchthat a specific binding between a polypeptide with an eptitopic regionon the amyloid protein may occur. The polypeptide/target complex mayconveniently be detected through a label attached to the polypeptide orany other art-known method of detection.

Further provided herein are kits for detection or diagnosis. Exemplarykits comprise one or more polypeptides provided herein along with one ormore reagents useful for detecting a complex between the one or morepolypeptides and their targets.

Preparation of Polypeptides Comprising a Modified Amino Acid Residues

The polypeptides described herein can be prepared by any techniqueapparent to those of skill in the art without limitation. Usefultechniques for preparation include in vivo synthesis, for example withmodified tRNA and tRNA synthetase, cell-free synthesis, for example withmodified tRNA and tRNA synthetase, solid phase polypeptide synthesis andliquid phase polypeptide synthesis. Exemplary techniques are describedin this section and in the examples herein. In particular embodiments,the polypeptide is an antibody or antibody fragment.

In certain embodiments, a variant of the aminoacyl tRNA synthetaseprovided in SEQ ID NO: 3 is used to catalyze the attachment of anon-natural amino acid to a compatible tRNA. Variants of the aminoacyltRNA synthetase of SEQ ID NO: 3 are particularly advantageous whenutilizing amino acids comprising tetrazine functional groups, such asthose provided in any of formulas I, Ia, II, III, IV, V, VI, VII, VIII,IX, and (1)-(10). In certain embodiments, a variant of SEQ ID NO: 3 withthe following mutations, designated “2A2”, may be particularlyadvantageous for use with a non-natural amino acid of formula 9: Y32L,L65V, H70A, F108W, Q109S, D158V, I159A, and L162V (SEQ ID NO: 4). Insome embodiments, a variant of SEQ ID NO: 3 with the followingmutations, designated “2A9”, may be particularly advantageous for usewith a non-natural amino acid of formula 6: Y32G, L65V, H70A, Q109S,D158G, and L162S (SEQ ID NO: 5). Other aminoacyl tRNA synthetases thatmay be useful with the compounds of the invention include the mtaFsynthetase disclosed in Seitchik et al., J. Am. Chem. Soc., 2012,134:2898-2901 (incorporated by reference in its entirety) and othervariants of SEQ ID NO: 3. Variants of SEQ ID NO: 3 may be made bymutagenesis and screened to identify mutant synthetases that act on anynon-natural amino acid of interest. Such mutagenesis may be completelyrandom, or may be deterministic with respect to the location of themutation(s) and/or the residue(s) allowed to occur at a particularportion of the synthetase polypeptide sequence. Examples of methods forrandom mutagenesis of synthetases may be found in Seitchik et al., citedabove and incorporated by reference in its entirety.

In certain methods, the polypeptide is translated and/or transcribedfrom one or more polynucleotides encoding the polypeptide. Accordingly,provided herein are polynucleotides capable of encoding the polypeptideshaving one or more modified amino acid residues at site-specificpositions in one or more polypeptide chains. In certain embodiments, thepolynucleotides comprise a codon not normally associated with an aminoacid at the polynucleotide position corresponding to the site-specificpolypeptide position for the modified amino acid residue. Examples ofsuch codons include stop codons, 4 bp codons, 5 bp codons, and the like.The reaction mixture typically comprises a tRNA synthetase capable ofmaking tRNAs that complement (suppress) said codon. These suppressortRNAs are linked to the modified amino acids to facilitate theirincorporation into the polypeptide at the site of the suppressor codon.

The polypeptides can be prepared by techniques known to those of skillin the art for expressing polynucleotides to incorporate modified aminoacid residues into site specific positions of a polypeptide. Suchtechniques are described, for example, in U.S. Pat. Nos. 7,045,337 and7,083,970, in U.S. Published Patent Application Nos. US 2008/0317670, US2009/0093405, US 2010/0093082, US 2010/0098630, US 2008/0085277 and ininternational patent publication nos. WO 2004/016778 A1 and WO2008/066583 A2, the contents of which are hereby incorporated byreference in their entireties.

In certain embodiments, a polypeptide can be prepared in a cell-freereaction mixture comprising at least one orthogonal tRNA aminoacylatedwith a modified amino acid residue, where the orthogonal tRNA base pairswith a codon that is not normally associated with an amino acid residue,e.g. a stop codon; a 4 bp codon, etc. The reaction mixture alsocomprises a tRNA synthetase capable of aminoacylating the orthogonaltRNA with a modified amino acid. One tRNA synthetase that can be used isshown as SEQ ID NO:55 and 56 in US Patent Publication No. 2008/0233611.Wild-type tyrosyl M janashcii tRNA may also be used. Usually theorthogonal tRNA synthetase, which is susceptible to degradation byproteases present in bacterial cell extracts, is exogenously synthesizedand added to the reaction mix prior to initiation of polypeptidesynthesis. The orthogonal tRNA may be synthesized in the bacterial cellsfrom which the cell extract is obtained, may be synthesized de novoduring the polypeptide synthesis reaction, or may be exogenously addedto the reaction mix.

In certain embodiments, components that affect modified amino acidresidue insertion and protein insertion or folding are optionally addedto the reaction mixture. Such components include elevated concentrationsof translation factors to minimize the effect of release factor 1 and 2and to further optimize orthogonal component concentrations. Proteinchaperones (Dsb System of oxidoreductases and isomerases, GroES, GroEL,DNAJ, DNAK, Skp, etc.) may be exogenously added to the reaction mixtureor may be overexpressed in the source cells used to prepare the cellextract The reactions may utilize a large scale reactor, small scale, ormay be multiplexed to perform a plurality of simultaneous syntheses.Continuous reactions will use a feed mechanism to introduce a flow ofreagents, and may isolate the end-product as part of the process. Batchsystems are also of interest, where additional reagents may beintroduced to prolong the period of time for active synthesis. A reactormay be run in any mode such as batch, extended batch, semi-batch,semi-continuous, fed-batch and continuous, and which will be selected inaccordance with the application purpose. The reactions may be of anyvolume, either in a small scale, usually at least about 1 μl and notmore than about 15 μl, or in a scaled up reaction, where the reactionvolume is at least about 15 μl, usually at least about 50 μl, moreusually at least about 100 μl, and may be 500 μl, 1000 μl, or greater.In principle, reactions may be conducted at any scale as long assufficient oxygen (or other electron acceptor) is supplied when needed.

Useful methods for synthesis where at least one modified amino acidresidue is introduced into the polypeptide strand during elongationinclude but are not limited to: (I) addition of exogenous purifiedorthogonal synthetase, modified amino acid, and orthogonal tRNA to thecell-free reaction, (II) addition of exogenous purified orthogonalsynthetase and modified amino acid to the reaction mixture, but withorthogonal tRNA transcribed during the cell-free reaction, (III)addition of exogenous purified orthogonal synthetase and modified aminoacid to the reaction mixture, but with orthogonal tRNA synthesized bythe cell extract source organism. In certain embodiments, the orthogonalcomponents are driven by regulatable promoters, so that synthesis levelscan be controlled although other measures may be used such ascontrolling the level of the relevant DNA templates by addition orspecific digestion.

In certain embodiments, tRNA synthetase is exogenously synthesized andadded to the cell-free reaction mix. In certain embodiments, thereaction mix is prepared from bacterial cells in which ompT has beeninactivated or is naturally inactive. OmpT is believed to degradecomponents of the reaction mixture including tRNA synthetase.

In addition to the above components such as cell-free extract, genetictemplate, and amino acids, materials specifically required for proteinsynthesis may be added to the reaction. These materials include salts,folinic acid, cyclic AMP, inhibitors for protein or nucleic aciddegrading enzymes, inhibitors or regulators of protein synthesis,adjusters of oxidation/reduction potential(s), non-denaturingsurfactants, buffer components, spermine, spermidine, putrescine, etc.

The salts preferably include potassium, magnesium, and ammonium salts(e.g. of acetic acid or glutamic acid). One or more of such salts mayhave an alternative amino acid as a counter anion. There is aninterdependence among ionic species for optimal concentration. Theseionic species are typically optimized with regard to protein production.When changing the concentration of a particular component of thereaction medium, that of another component may be changed accordingly.For example, the concentrations of several components such asnucleotides and energy source compounds may be simultaneously adjustedin accordance with the change in those of other components. Also, theconcentration levels of components in the reactor may be varied overtime. The adjuster of oxidation/reduction potential may bedithiothreitol, ascorbic acid, glutathione and/or their oxidized forms.

In certain embodiments, the reaction can proceed in a dialysis mode, ina diafiltration batch mode, in a fed-batch mode of in a semi-continuousoperation mode. In certain embodiments, a feed solution can be suppliedto the reactor through a membrane or through an injection unit.Synthesized polypeptide can accumulate in the reactor followed byisolation or purification after completion of the system operation.Vesicles containing the polypeptide may also be continuously isolated,for example by affinity adsorption from the reaction mixture either insitu or in a circulation loop as the reaction fluid is pumped past theadsorption matrix.

During protein synthesis in the reactor, the protein isolating means forselectively isolating the desired protein may include a unit packed withparticles coated with polypeptide molecules or other molecules foradsorbing the synthesized, desired protein. Preferably, the proteinisolating means comprises two columns for alternating use.

The resulting polypeptide can be purified or isolated by standardtechniques. Exemplary techniques are provided in the examples herein.

Assay Methods

Polypeptides can be assayed for their expected activity, or for a newactivity, according to any assay apparent to those of skill in the art.The resulting polypeptide can be assayed for activity in a functionalassay or by quantitating the amount of protein present in anon-functional assay, e.g. immunostaining, ELISA, quantitation onCoomasie or silver stained gel, etc., and determining the ratio ofbiologically active protein to total protein.

The amount of protein produced in a translation reaction can be measuredin various fashions. One method relies on the availability of an assaywhich measures the activity of the particular protein being translated.An example of an assay for measuring protein activity is a luciferaseassay system, or chloramphenicol acetyl transferase assay system. Theseassays measure the amount of functionally active protein produced fromthe translation reaction. Activity assays will not measure full lengthprotein that is inactive due to improper protein folding or lack ofother post translational modifications necessary for protein activity.

Another method of measuring the amount of protein produced in coupled invitro transcription and translation reactions is to perform thereactions using a known quantity of radiolabeled amino acid such as³⁵S-methionine, ³H-leucine or ¹⁴C-leucine and subsequently measuring theamount of radiolabeled amino acid residue incorporated into the newlytranslated protein. Incorporation assays will measure the amount ofradiolabeled amino acid residues in all proteins produced in an in vitrotranslation reaction including truncated protein products. Theradiolabeled protein may be further separated on a protein gel, and byautoradiography confirmed that the product is the proper size and thatsecondary protein products have not been produced.

Preparation of Modified Amino Acids

The compounds provided herein can be prepared, isolated or obtained byany method apparent to those of skill in the art. Compounds providedherein can be prepared according to the General Preparation Schemeprovided herein. Reaction conditions, steps and reactants not providedin the General Preparation Scheme would be apparent to, and known by,those skilled in the art. Exemplary methods of making the compounds areprovided in the Examples below.

General Preparation Scheme 1

EXAMPLES

As used herein, the symbols and conventions used in these processes,schemes and examples, regardless of whether a particular abbreviation isspecifically defined, are consistent with those used in the contemporaryscientific literature, for example, the Journal of Biological Chemistry.

For all of the following examples, standard work-up and purificationmethods known to those skilled in the art can be utilized. Unlessotherwise indicated, all temperatures are expressed in ° C. (degreesCentigrade). All methods are conducted at room temperature unlessotherwise noted.

Example 1: Preparation of the Compound of Formula (6)

The compound of formula (6) was prepared according to the followingscheme and protocols.

Preparation of Compound 1

To a suspension of zinc powder (1.37 g, 21 mmol, 2.0 eq) in DMF at roomtemperature under N₂ was added iodine (400 mg, 1.08 mmol, 0.15 eq). Thesuspension was stirred at room temperature for about 10 minutes whilethe color changed from brown to grey. To the suspension was addedcarbamic acid, N-(3-iodopropyl)-1,1-dimethylethyl ester (3.45 g, 10.5mmol, 1.0 eq) along with iodine (400 mg, 1.08 mmol, 0.15 eq). Thereaction mixture was allowed to stir at room temperature for 30 minutes(heat produced from the reaction can be observed) and cooled to roomtemperature to give a crude zinc reagent, which was used for the nextstep without further purification.

A mixture of the 2-aminyl-5-bromopyridine (2.2 g, 12.6 mmol, 1.2 eq),Pd₂(dba)₃ (292 mg, 0.318 mmol, 0.025 eq) and2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl, SPhos (0.52 g, 1.27mmol, 0.1 eq) in a Schlenk tube in anhydrous DMF (5 mL) under N₂ wasstirred at room temperature for 5 min. The above prepared zinc reagent(1.0 eq) suspension was added into the mixture under N₂. The reactionwas heated to 60° C. for 6 hours. It was cooled to room temperature andfiltered through a pad of Celite. The filtrate was concentrated andpurified by a silica gel column (DCM:MeOH=9:1) to give compound 1 and2-aminyl-5-bromopyridine (could not be separated at this stage).

Preparation of Compound 2

To a mixture of compound 1 (containing 2-aminyl-5-bromopyridine) in amixed solvent of THF (5 mL) and MeOH (10 mL) was added aqueous LiOH (800mg, 19 mmol, 5.0 eq, in 10 mL of water). The mixture was stirred at roomtemperature for 2 hours. The solvent was removed and the residue waspurified by a silica gel column (DCM:MeOH:Et₃N, 9:1:1) to give product 2(1.4 g, 37% two-steps) as a triethylamine salt.

Preparation of Compound 3

To a triethylamine salt of 2 (1.4 g, 3.66 mmol, 1.0 eq) in DMF (10.0 mL)was added 4-methyltetrazine methyl sulfide (1.56 g, 11.0 mmol, 3.0 eq)in a sealed tube. The tube was heated to 100° C. for 60 hours. Thereaction mixture was cooled and purified by prep-HPLC to give product 3(480 mg, 35%).

Preparation of the Compound Offormula (6)

Compound 3 (190 mg, 0.51 mmol) was dissolved in 20% TFA in DCM (8 mL)and stirred at room temperature for 1 hour. The solvent was removedunder reduced pressure, and the residue was re-dissolved in water,frozen and lyophilized to give a compound of formula (6) (240 mg, 78%,TFA salts) as red solid. LC-MS (ESI): 276 (M+1), 274 (M−1). ¹H NMR (300MHz, CD₃OD) δ 8.29 (s, 1H), 8.04 (d, J=7.8 Hz, 1H), 7.91 (dd, J=7.8 Hz,2.4 Hz, 1H), 4.33 (t, 1H), 3.25-3.34 (m, 2H), 2.91 (s, 3H).

Example 2: Preparation of the Compound of Formula (31

Preparation of Compound 4

To a suspension of zinc powder (392 mg, 6.0 mmol, 2.0 eq) in DMF (1 mL)at room temperature under N₂ was added iodine (114 mg, 0.45 mmol, 0.15eq). The suspension was stirred at room temperature for about 10 minuteswhile the color changed from brown to grey. To the suspension was addedcarbamic acid, N-(3-iodopropyl)-1,1-dimethylethyl ester (987 mg, 3.0mmol, 1.0 eq) along with iodine (114 mg, 0.45 mmol, 0.15 eq). Thereaction mixture was allowed to stir at room temperature for 30 minutes(heat produced from the reaction can be observed) and cooled to roomtemperature to give a crude zinc reagent, which was used for the nextstep without further purification.

A mixture of the 3-aminyl-5-bromopyridine (623 mg, 3.6 mmol, 1.2 eq),Pd₂(dba)₃ (69 mg, 0.075 mmol, 0.025 eq) and ligand SPhos (123 mg, 0.3mmol, 0.1 eq) in Schlenk tube was added anhydrous DMF (2 mL) under N₂.The mixture was stirred at rt for 5 min. The above prepared zinc reagent(1.0 eq) suspension was added into the mixture under N₂. The reactionwas heated to 50° C. for 5 hours. It was cooled to room temperature andfiltered through a pad of Celite. The filtrate was concentrated andpurified by a silica gel column (DCM:MeOH=9:1) to give compound 4.

Preparation of Compound 5

To a mixture of compound 4 (containing 3-aminyl-5-bromopyridine) in amixed solvent of THF (2 mL) and MeOH (5 mL) was added aqueous lithiumhydroxide (198 mg, 4.7 mmol, 2.0 eq, in 5 mL of water). The mixture wasstirred at rt for 2 h. The solvent was removed and the residue waspurified by C₁₈ column (MeOH: H₂O) to give product 5 (480 mg, 57% yield,two-steps).

Preparation of Compound 6

To a solution of compound 5 (281 mg, 1.0 mmol, 1.0 eq) in THF (5 mL) wasadded 4-methyl tetrazine methyl sulfoxide (316 mg, 2.0 mmol, 2.0 eq,freshly prepared from its sulfide by oxidation with mCPBA) in a sealedtube. The tube was heated at 80° C. for 48 h. The solvent was removedand product was purified by prep-HPLC to give product 6 (30 mg, 8%).

Preparation of the Compound of Formula (3)

Compound 6 (30 mg) was dissolved in 20% TFA in DCM (5 mL) and stirred atrt for 1 h. The solvent was removed under reduced pressure and theresidue was re-dissolved in water, frozen and lyophilized to give acompound of formula (3) (25 mg, TFA salts) as red solid. LC-MS (ESI):276 (M+1), 274 (M−1). ¹H NMR (300 MHz, D₂O) δ 9.18 (s, 1H), 8.56 (s,1H), 8.35 (s, 1H), 4.33 (t, 1H), 3.30-3.40 (m, 2H), 2.80 (s, 3H).

Example 3: Preparation of the Compound of Formula (9)

Preparation of Compound 7

To a suspension of zinc powder (130 mg, 2 mmol, 2.0 eq) in DMF at rtunder N₂ was added iodine (38 mg, 0.15 mmol, 0.15 eq). The suspensionstirred at rt about 10 min while the color changed from brown to grey.To the suspension was added carbamic acid,N-(3-iodopropyl)-1,1-dimethylethyl ester (329 mg, 1 mmol, 1.0 eq) alongwith iodine (38 mg, 0.15 mmol, 0.15 eq). The reaction mixture allowed tostir at rt for 30 min (heat produced from the reaction can be observed)and cooled to rt to give a crude zinc reagent, which was used for nextstep without further purification.

A mixture of the 3-cyano-6-bromopyridine (183 mg, 1 mmol, 1.0 eq),Pd₂(dba)₃ (23 mg, 0.025 mmol, 0.025 eq) and ligand SPhos (41 mg, 0.1mmol, 0.1 eq) in anhydrous DMF (2 mL) under N₂ in a Schlenk tube wasstirred at rt for 5 min. The above prepared zinc reagent (1.0 eq)suspension was added into the mixture under N₂. The reaction was heatedto 60° C. for 6 h. It was cooled to rt and filtered through a pad ofCelite. The filtrate was concentrated and purified by silica gel column(Hexanes:ethyl acetate=1:1) to give compound 7 (145 mg, 45%).

Preparation of Compound 8

To compound 7 (185 mg, 0.61 mmol, 1.0 eq) in dry MeOH (5 mL) at 0° C.was added NiCl₂.6H₂O (71 mg, 0.3 mmol, 0.5 eq), and NaBH₄ (229 mg, 6.1mmol, 10 eq) in portions. The mixture was stirred at rt for 30 min. Thereaction mixture was filtered through a pad of Celite, and concentratedto give a crude product, which was purified by silica gel column(DCM:MeOH:Et₃N, 9:1:1) to give compound 8 (190 mg).

Preparation of Compound 9

To a solution of compound 8 (1.5 g, 5.1 mmol, 1.0 eq) in a mixed solventof THF (5 mL) and MeOH (10 mL) was added aqueous LiOH (427 mg, 10.2mmol, 2.0 eq, in 10 mL of water). The mixture was stirred at rt for 2 h.The reaction mixture was concentrated to a small volume and most lithiumsalts were precipitated out and removed by filtration. The filtrate wasconcentrated to give product 9 (1.6 g, still containing some lilhiuiiisalt).

Preparation of Compound 10

To a product 9 (800 mg, 2.85 mmol, 1.0 eq) in MeOH (10.0 mL) was added4-methyltetrazine methyl sulfide (808 mg, 5.7 mmol, 2.0 eq) andtriethylamine (0.792 mL, 5.7 mmol, 2.0 eq) in a sealed tube. The tubewas heated to 80° C. for 72 h. The reaction mixture was cooled andpurified by prep-HPLC to give product 10 (460 mg, 43%).

Preparation of Compound a Compound of Formula (9)

Compound 10 (470 mg, 1.21 mmol) was dissolved in 20% TFA in DCM (8 mL)and stirred at rt for 1 h. The solvent was removed under reducedpressure, and the residue was re-dissolved in water, frozen andlyophilized to give a compound of formula (9) (660 mg, TFA salts) as redsolid. LC-MS (ESI): 290 (M+1), 288 (M−1). ¹H NMR (300 MHz, CD₃OD) δ 8.61(d, =1.8 Hz, 1H), 7.93 (dd, J=2.4 and 8.1 Hz, 1H), 7.42 (d, J=8.1 Hz,1H), 4.73 (s, 2H), 4.44 (m, 1H), 3.35-3.50 (m, 2H), 2.74 (s, 3H).

Example 4: Preparation of Green Fluorescent Protein Comprising aCompound of Formula 9 or a Compound of Formula 6

This example provides two novel tRNA synthetases useful withtetrazine-containing amino acids described herein. These twosynthetases, designated “2A2” (SEQ ID NO: 4) and “2A9” (SEQ ID NO: 5)were produced by mutagenesis of wild-type tyrosyl-tRNA synthetase(TyrRS) from Methanococcus jannaschii (SEQ ID NO:3). Mutagenesis wasperformed by assembling overlapping oligonucleotides encoding themutations by polymerase chain reaction (PCR). Mutants were screenedutilizing the green fluorescent protein expression assay describedbelow.

The ability of the novel tRNA synthetases to incorporate modified aminoacids into a polypeptide was evaluated in cell-free protein synthesisreactions comprising DNA encoding SUPERFOLDER green fluorescent protein(GFP). The codon encoding position 49 of the GFP polypeptide, whichnormally encodes a lysine residue, was substituted with an amber stopcodon (TAG). Full-length GFP is expressed only if the novel tRNAsynthetases incorporate s modified amino acid into the polypeptidechain. The level of expression of full-length GFP can be evaluated bymeasuring fluorescence.

The GFP DNA containing the TAG stop codon was expressed in cell-freeprotein synthesis reactions as described in Zawada et al., 2011,Biotechnol. Bioeng. 108(7)1570-1578 with the following modifications:the cell-free extracts were prepared from an OmpT sensitive RF-1attenuated strain that was also engineered to overexpress E. coli DsbC,and a similar RF-1 attenuated E. coli strain that was engineered toproduce an orthogonal CUA-encoding tRNA for insertion of a non-naturalamino acid at an amber stop codon. The cell-free extracts were blended(at a ratio of 85:15, respectively), and then added to a premixcontaining all other components of a cell-free protein synthesis systemexcept for DNA encoding GFP (K49TAG).

The final cell-free protein synthesis reactions contained 30% cellextract, with or without 2 mM non-natural amino acid (either a compoundof formula 6 or a compound of formula 9), a novel tRNA synthetase (2A2or 2A9 at a concentration of 4 μM, 2 uM, 1 uM or zero), 8 mM magnesiumglutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 35 mMsodium pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mMamino acids (except 0.5 mM for tyrosine and phenylalanine), 4 mM sodiumoxalate, 1 mM putrescine, 1.5 mM spermidine, 15 mM potassium phosphate,100 nM T7 RNA polymerase, and 10 μg/mL GFP (K49TAG) expression templateplasmid. The cell-free synthesis reactions were initiated by theaddition of the plasmid DNA encoding GFP (K49TAG). The reactions wereincubated at 30° C. for 12 h on a shaker at 650 rpm. After 12 hours ofexpression, the GFP fluorescence signal was measured at an excitationwavelength of 476 nm, and an emission wavelength of 510 nm, on aSPECTRAMAX M5 fluorescence plate reader.

FIG. 1 shows the results for the combination of tRNA synthetase 2A2without a compound of formula 9 and with a compound of formula 9 at 4mM. At a 4 μM concentration of 2A2, the amount of fluorescence isapproximately equivalent to that of native GFP (“GFP”) positive control,indicating that GFP comprising a compound of formula 9 is produced atapproximately the same level as native GFP (without K49TAG). In FIG. 1,the y-axis shows fluorescence, in relative fluorescence units (RFU);“aaRS” is 2A2; “GFP” is native GFP (without K49TAG); and “negativecontrol” is a cell-free protein synthesis reaction without a DNAtemplate.

FIG. 2 shows the results for the combination of tRNA synthetase 2A9without a compound of formula 6 and with a compound of formula 6 at 1mM. At a 2-4 μM concentration of 2A9, the amount of fluorescenceapproaches that of native GFP (“GFP”) positive control, indicating thatGFP comprising a compound of formula 6 is produced at a levelapproaching that of native GFP (without K49TAG). In FIG. 2, the y-axisshows fluorescence, in relative fluorescence units (RFU); “aaRS” is 2A9;“GFP” is native GFP (without K49TAG); and “negative control” is acell-free protein synthesis reaction without a DNA template.

Example 5: Solubility of Selected Non-Natural Amino Acids ComprisingTetrazine Functionality

This example describes the solubility of three non-natural amino acidscomprising tetrazine functional groups in phosphate buffered salinesolution, with a pH of 7.4 (PBS). The example also illustrates anddiscusses the useful increased solubility of the pyridyl tetrazine aminoacids provided herein when compared to a phenyl tetrazine-comprisingnon-natural amino acid of the art.

The three compounds tested were AB 4091, compound (9), and compound (6).The structures of these compounds are provided below:

As described below, AB 4091 has poor aqueous solubility. To address thisissue, novel pyridyl tetrazine compounds (e.g., compound (9) andcompound (6)) were designed and synthesized. In the novel compounds, thephenyl moiety of AB 4091 was replaced with a pyridine group, to improvethe molecular surface polarity and to reduce the lattice energy of thesolid form, while conserving the structure of the compound forrecognition by the appropriate tRNA during tRNA charging.

Solubility of AB 4091 at Room Temperature

A suspension of AB 4091 was formed by adding 1.8 mg of AB 4091 to 5 mLof PBS. The suspension was vortexed for five minutes and sonicated fortwo minutes. Suspended particles remained. Additional PBS was added tothe suspension in small portions, with vortexing, to reach a totalvolume of 25 mL of PBS. This mixture remained cloudy after shakingovernight at room temperature and turned clear following the addition of2 mL of PBS with vortexing. Clarity of the solution was determined byvisual inspection. The solubility of AB 4091 is about 215 μM at roomtemperature.

Solubility of Compound (9) at Room Temperature

A solution of compound (9) was formed by adding 8.3 mg of compound (9)to 200 of PBS to form a homogeneous solution. An additional 11.5 mg ofcompound (9) was added to this solution, in small portions, over aperiod of 20 minutes, with vortexing. The solution remained clear aftervortexing, with no particulates or cloudiness observed. The maximumsolubility of compound (9) was not reached, due to its high solubilityand the limited amount of the compound available. The finalconcentration achieved was 342 mM at room temperature, over 1600-foldgreater than the solubility of AB 4091.

Solubility of Compound (6) at Room Temperature

A solution of compound (6) was formed by adding 8.7 mg of compound (6)to 200 μL of PBS to form a homogenous solution. An additional 26.9 mg ofcompound (6) was added to this solution, in small portions, over aperiod of 25 minutes, with vortexing. The solution remained clear aftervortexing, with no particulates or cloudiness observed. The maximumsolubility of compound (6) was not reached, due to its high solubilityand the limited amount of the compound available. The finalconcentration achieved was 647 mM at room temperature, over 3000-foldgreater than the solubility of AB 4091.

Pyridyl Compounds Exhibit Greater than Expected Solubility

The calculated log P (c Log P) value is a calculated measure of thepartition coefficient of a compound between n-octanol and water. Thevalue is a generally accepted measure of a compound's hydrophilicity,with lower c Log P values indicating higher solubility in aqueous media.This correlation is illustrated by the empirical equation Log S=0.5-logP-0.01(TM-25), where S is the solubility and TM is the melting point ofthe molecule and an indicator of lattice energy of the solid.

The c Log P value was calculated for compounds AB 4091, (9), and (6),according to the methods described in Leo AJ, “Methods of calculatingpartition coefficients,” in Comprehensive Medicinal Chemistry: TheRational Design, Mechanistic Study & Therapeutic Application of ChemicalCompounds, volume 4. New York: Pergamon Press. Chapter 18.7, pp.295-617, incorporated by reference in its entirety, as implemented inChemDraw software.

The c Log P values were 1.68, 0.36, and 1.06 for AB 4091, compound (9),and compound (6), respectively. The moderate reduction in the c Log Pvalues alone, between AB 4091 and the pyridyl compounds, does notexplain the significant increase in solubility observed in the pyridylcompounds (over 1600-fold for compound (9) and over 3000-fold forcompound (6)).

Without being bound by theory, it is possible that the nitrogensubstitution in the pyridyl compounds influences their solid-statestability by changing their crystal lattice energy. The fact that AB4091 is a solid powder at room temperature, while compound (6) is anoil, suggests a difference in the crystal lattice energy of the twocompounds. Moreover, the pyridyl moiety is capable of forming hydrogenbonds with water upon solvation, while the phenyl moiety does not. Thesefactors, in addition to the changes in surface polarity realized byintroducing the nitrogen, may contribute to the enhanced solubility ofthe pyridyl compounds provided herein.

The novel pyridyl tetrazine amino acids provided herein are particularlyuseful when incorporated into proteins in a cell-free protein synthesisreaction. Prior compounds, i.e., the phenyl tetrazine amino acids suchas AB 4091, were very difficult to use in such reactions because oftheir poor solubility. Thus, the reactions required very carefulintroduction of the phenyl tetrazine compounds into the cell-freeprotein synthesis reaction so that the amino acid did not immediatelyprecipitate, and further required extensive vortexing to re-dissolve anyprecipitated material. Without taking such care, the effectiveconcentration of the amino acid in the cell-free protein synthesisreaction would be much lower than expected, which then would result in alow concentration of tRNA charged with the amino acid, leading to poorsuppression of the amber codon and premature termination of proteinsynthesis, which ultimately results in poor yields of full length,properly folded protein containing the desired amino acid. In addition,the lower than expected phenyl tetrazine concentration can also lead toincreased misincorporation of wild type amino acids instead of thedesired phenyl tetrazine at the amber codon site.

In contrast, the novel pyridyl tetrazine compounds provided hereinreadily mix into the cell-free protein synthesis reaction mixturewithout precipitation and reliably yield full length properly-foldedprotein products without demonstrating excessive premature terminationof protein synthesis or misincorporation of undesired wild type aminoacids.

Example 6: Incorporation of Tetrazine and Azide Functional Groups intoan IgG Antibody

This example demonstrates the incorporation of two non-natural aminoacids (nnAAs) into a trastuzumab parent antibody. The nnAAs wereincorporated into the antibody by placing an amber codon at the desiredposition for nnAA incorporation in a plasmid encoding the trastuzumabheavy chain and in a plasmid encoding a trastuzumab light chain. nnAA1,para-azido-methyl phenylalanine (pAMF) was incorporated at position S7of the trastuzumab light chain. AB 4091, described above, wasincorporated at position F404 of the trastuzumab heavy chain.

Both the heavy chain and the light chain were synthesized by cell-freeprotein synthesis. The light chain was synthesized first. The cell-freereaction mix comprised an 85%:15% blend of cell-free extracts made froman OmpT sensitive RF-1 attenuated E. coli strain that was engineered tooverexpress DsbC (DsbC extract), and an OmpT sensitive RF-1 attenuatedE. coli strain which was engineered to produce an orthogonalCUA-encoding tRNA (tRNA extract) for insertion of a non-natural aminoacid at an amber stop codon. Cell-free extracts were treated with 50 μMiodoacetamide for 30 min at RT (20° C.) and added to a premix containingall other components except for DNA encoding the light chain variant.The final concentration in the protein synthesis reaction was 30% cellextract, 2 mM pAMF (RSP Amino Acids), 0.37 mg/mL pAzMeF-specificamino-acyl tRNA synthetase (FRS), 2 mM oxidized glutathione (GSSG), 8 mMmagnesium glutamate, 10 mM ammonium glutamate, 130 mM potassiumglutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP,and CMP, 2 mM amino acids (except 0.5 mM for tyrosine andphenylalanine), 4 mM sodium oxalate, 1 mM putrescine, 1.5 mM spermidine,15 mM potassium phosphate, 100 nM T7 RNA polymerase, and 10 μg/mLtrastuzumab light chain variant DNA. The FRS was thep-cyanophenylalanine-specific aminoacyl tRNA synthetase described inYoung et al., Biochem., 2011, 50:1894-1900, incorporated by reference inits entirety. After addition of the DNA template, cell-free reactionswere incubated at 30° C. for 12 h. The synthesized light chains werepurified with a protein L column.

The second nnAA, AB 4091, was incorporated into the heavy chain in thepresence of prefabricated light chain containing pAMF, using the samereaction conditions described above, with the following changes: thennAA was changed to AB 4091; the synthetase was changed to an AB4091-specific aminoacyl tRNA synthetase; and the reaction was performedin the presence of 10 μg/mL of DNA encoding the trastuzumab heavy chainwith an amber stop codon at position F404 and 500 μg/mL of prefabricatedLC. The aminoacyl tRNA synthetase used in this reaction was the mtaFsynthetase disclosed in Seitchik et al., J. Am. Chem. Soc., 2012,134:2898-2901, incorporated by reference in its entirety.Autoradiography (FIG. 3) suggests that about 1 mg/mL IgG containing twonnAAs was produced.

This example demonstrates production of assembled IgG having pAMF in thelight chain and a tetrazine-containing amino acid (AB 4091) in the heavychain. Alternatively, one can incorporate a tetrazine-containing aminoacid in the light chain and an azide-containing amino acid in the heavychain by substituting the appropriate amino acid and aminoacyl tRNAsynthetase during transcription and translation of each template DNA.Additionally, this approach can be used with any of the nnAAs known inthe art or described herein. One of ordinary skill in the art will alsorecognize that incorporation of multiple nnAAs may be achieved in asingle reaction by utilizing orthogonal tRNAs, as described below.

Example 7: Simultaneous Site-Specific Incorporation of Two DifferentNon-Natural Amino Acids, Enabling Mutually Orthogonal ConjugationChemistries

This example describes the site-specific incorporation of differentnon-natural amino acid residues into antibodies in a single reactionmixture, and the subsequent mutually orthogonal conjugation of drugs tothese non-natural amino acids.

Cell-free protein synthesis reactions were carried out essentially asdescribed in Example 6, with the following variations to enablesite-specific incorporation of nnAAs using two different stop codons.Aminoacyl tRNA synthetases PyrTetRS (SEQ ID NO: 6) and PyIRS (SEQ ID NO:7) were expressed and purified separately and added as exogenouscomponents to the cell free expression reactions. Reactions alsocontained orthogonal suppressor tRNAs recognized exclusively by PyrTetRSor Py1RS. In this embodiment, PyrTetRS charges an opal codon (TGA)suppressor tRNA (SEQ 1D NO: 8) with compound (6) and Py1RS charges anamber codon (TAG) suppressor tRNA (SEQ ID NO: 9) with compound A19 andthe ochre codon (TAA) is used to terminate protein translation. PlasmidDNA templates encoding product proteins possessed TAG or TGA codons atthe codon position corresponding to the desired site of nnAAincorporation. This may include incorporation of two different nnAAsinto a single polypeptide (e.g., the heavy chain or the light chain) orinto different polypeptides (e.g., one compound on the heavy chain andone compound on the light chain).

Non-natural amino acid compounds were incorporated into the heavy andlight chains of three antibodies: trastuzumab (HC: SEQ ID NO: 1; LC: SEQID NO: 2); brentuximab (HC: SEQ ID NO: 10; LC: SEQ ID NO: 11); andanti-CD74 (HC: SEQ ID NO: 12; LC: SEQ ID NO: 13). These antibodies werethen conjugated to either DBCO-Gemcitabine, or DBCO-DM1 and to TCO-MMAFin a single pot reaction under the essentially same reaction conditionsas described in Example 6. The chemical structures of DBCO-Gemcitabineand TCO-MMAF are provided below.

Drug-to-antibody (DAR) ratios were determined by liquid chromatographymass spectrometry.

Samples were run on a Waters Aquity UPLC system attached a Xevo QTOF.Proteins were separated on an Agilent PLRP-S column (2.3×50 mm, 5 μm,4000 Å) at 80° C. Mobile phases: A: 0.1% formic acid water; B: 20:80isopropanol:acetonitrile, 0.1% formic acid. Samples were desalted oncolumn for 0.4 minutes at 10% B followed by a step gradient from 30% Bto 40% B over 7 minutes, 40% B to 60% B over 3 minutes. Data wasacquired over the whole LC elution using a cone voltage of 35V. Spectrawere analyzed using MassLynx software. DAR values were calculated as aweighted average using the peak intensity of the matching peaks in thedeconvoluted spectra. Where a defined peak was not observed the baselineintensity at the theoretical mass of the conjugate was used in the DARcalculation.

Table 4 shows the resulting DARs for various combinations of antibody,sites of incorporation of nnAAs, and drugs conjugated to theincorporated nnAAs.

TABLE 10 Drug-to-Antibody Ratios for Antibodies Produced in a SingleReaction Mixture Antibody nnAA Site 1 nnAA Site 2 Drug 1 Drug 2 DARTrastuzumab HC S136 AEK LC S7 PyrTet DBCO- TCO-MMAF 2.6 GemcitabineTrastuzumab HC K147 AEK LC S7 PyrTet DBCO-DM1 TCO-MMAF 3.3 TrastuzumabHC S25 PyrTet LC K45 AEK TCO-MMAF DBCO-DM1 3.6 Trastuzumab HC 525 PyrTetHC K147 AEK TCO-MMAF DBCO-DM1 3.0 Trastuzumab LC S7 PyrTet LC K39 AEKTCO-MMAF DBCO-DM1F 1.8 Trastuzumab LC S7 PyrTet LC S77 AEK TCO-MMAFDBCO-DM1 2.6 Trastuzumab LC S7 PyrTet LC R142 AEK TCO-MMAF DBCO-DM1 2.6Brentuximab HC K147 AEK LC S7 PyrTet DBCO-DM1 TCO-MMAF 2.7 BrentuximabLC S27 PyrTet LC R147 AEK TCO-MMAF DBCO-DM1 1.6 Brentuximab HC S25PyrTet HC K147 AEK TCO-MMAF DBCO-DM1 2.1 Anti-CD74 HC K147 AEK LC S7PyrTet DBCO-DM1 TCO-MMAF 2.0 Anti-CD74 LC S7 PyrTet LC R143 AEK TCO-MMAFDBCO-DM1 2.8 Anti-CD74 HC S25 PyrTet HC K147 AEK TCO-MMAF DBCO-DM1 2.0PyrTet = compound of formula (A6); AEK = compound of formula (A19); DAR= total drug-to-antibody ratio (scale: 0 to 4).

In all instances, species were identified in the deconvoluted massspectra that corresponded to an IgG conjugated to both drugssimultaneously in ratios of 1:1, 2:1, 1:2, and/or 2:2 indicatingsuccessful incorporation of both nnAAs and simultaneous conjugation toat least one molecule of each drug.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. While the claimed subject matter has beendescribed in terms of various embodiments, the skilled artisan willappreciate that various modifications, substitutions, omissions, andchanges may be made without departing from the spirit thereof.Accordingly, it is intended that the scope of the subject matter limitedsolely by the scope of the following claims, including equivalentsthereof

TABLE 1 Positions in SEQ ID NOs: 1 (heavy chain) and 2 (light chain) andcorresponding Kabat, Chothia, and EU residue numbers. HEAVY CHAIN LIGHTCHAIN Pos. in Pos. in SEQ ID SEQ ID NO: 1 Residue Kabat Chothia EU NO: 2Residue Kabat Chothia EU 1 E H1 E H1 E 1 D L1 D L1 D 2 V H2 V H2 V 2 IL2 I L2 I 3 Q H3 Q H3 Q 3 Q L3 Q L3 Q 4 L H4 L H4 L 4 M L4 M L4 M 5 V H5V H5 V 5 T L5 T L5 T 6 E H6 E H6 E 6 Q L6 Q L6 Q 7 S H7 S H7 S 7 S L7 SL7 S 8 G H8 G H8 G 8 P L8 P L8 P 9 G H9 G H9 G 9 S L9 S L9 S 10 G H10 GH10 G 10 S L10 S L10 S 11 L H11 L H11 L 11 L L11 L L11 L 12 V H12 V H12V 12 S L12 S L12 S 13 Q H13 Q H13 Q 13 A L13 A L13 A 14 P H14 P H14 P 14S L14 S L14 S 15 G H15 G H15 G 15 V L15 V L15 V 16 G H16 G H16 G 16 GL16 G L16 G 17 S H17 S H17 S 17 D L17 D L17 D 18 L H18 L H18 L 18 R L18R L18 R 19 R H19 R H19 R 19 V L19 V L19 V 20 L H20 L H20 L 20 T L20 TL20 T 21 S H21 S H21 S 21 I L21 I L21 I 22 C H22 C H22 C 22 T L22 T L22T 23 A H23 A H23 A 23 C L23 C L23 C 24 A H24 A H24 A 24 R L24 R L24 R 25S H25 S H25 S 25 A L25 A L25 A 26 G H26 G H26 G 26 S L26 S L26 S 27 FH27 F H27 F 27 Q L27 Q L27 Q 28 N H28 N H28 N 28 D L28 D L28 D 29 I H29I H29 I 29 V L29 V L29 V 30 K H30 K H30 K 30 N L30 N L30 N 31 D H31 DH31 D 31 T L31 T L31 T 32 T H32 T H32 T 32 A L32 A L32 A 33 Y H33 Y H33Y 33 V L33 V L33 V 34 I H34 I H34 I 34 A L34 A L34 A 35 H H35 H H35 H 35W L35 W L35 W 36 W H36 W H36 W 36 Y L36 Y L36 Y 37 V H37 V H37 V 37 QL37 Q L37 Q 38 R H38 R H38 R 38 Q L38 Q L38 Q 39 Q H39 Q H39 Q 39 K L39K L39 K 40 A H40 A H40 A 40 P L40 P L40 P 41 P H41 P H41 P 41 G L41 GL41 G 42 G H42 G H42 G 42 K L42 K L42 K 43 K H43 K H43 K 43 A L43 A L43A 44 G H44 G H44 G 44 P L44 P L44 P 45 L H45 L H45 L 45 K L45 K L45 K 46E H46 E H46 E 46 L L46 L L46 L 47 W H47 W H47 W 47 L L47 L L47 L 48 VH48 V H48 V 48 I L48 I L48 I 49 A H49 A H49 A 49 Y L49 Y L49 Y 50 R H50R H50 R 50 S L50 S L50 S 51 I H51 I H51 I 51 A L51 A L51 A 52 Y H52 YH52 Y 52 S L52 S L52 S 53 P H52A P H52A P 53 F L53 F L53 F 54 T H53 TH53 T 54 L L54 L L54 L 55 N H54 N H54 N 55 Y L55 Y L55 Y 56 G H55 G H55G 56 S L56 S L56 S 57 Y H56 Y H56 Y 57 G L57 G L57 G 58 T H57 T H57 T 58V L58 V L58 V 59 R H58 R H58 R 59 P L59 P L59 P 60 Y H59 Y H59 Y 60 SL60 S L60 S 61 A H60 A H60 A 61 R L61 R L61 R 62 D H61 D H61 D 62 F L62F L62 F 63 S H62 S H62 S 63 S L63 S L63 S 64 V H63 V H63 V 64 G L64 GL64 G 65 K H64 K H64 K 65 S L65 S L65 S 66 G H65 G H65 G 66 R L66 R L66R 67 R H66 R H66 R 67 S L67 S L67 S 68 F H67 F H67 F 68 G L68 G L68 G 69T H68 T H68 T 69 T L69 T L69 T 70 I H69 I H69 I 70 D L70 D L70 D 71 SH70 S H70 S 71 F L71 F L71 F 72 A H71 A H71 A 72 T L72 T L72 T 73 D H72D H72 D 73 L L73 L L73 L 74 T H73 T H73 T 74 T L74 T L74 T 75 S H74 SH74 S 75 I L75 I L75 I 76 K H75 K H75 K 76 S L76 S L76 S 77 N H76 N H76N 77 S L77 S L77 S 78 T H77 T H77 T 78 L L78 L L78 L 79 A H78 A H78 A 79Q L79 Q L79 Q 80 Y H79 Y H79 Y 80 p L80 P L80 P 81 L H80 L H80 L 81 EL81 E L81 E 82 Q H81 Q H81 Q 82 D L82 D L82 D 83 M H82 M H82 M 83 F L83F L83 F 84 N H82A N H82A N 84 A L84 A L84 A 85 S H82B S H82B S 85 T L85T L85 T 86 L H82C L H82C L 86 Y L86 Y L86 Y 87 R H83 R H83 R 87 Y L87 YL87 Y 88 A H84 A H84 A 88 C L88 C L88 C 89 E H85 E H85 E 89 Q L89 Q L89Q 90 D H86 D H86 D 90 Q L90 Q L90 Q 91 T H87 T H87 T 91 H L91 H L91 H 92A H88 A H88 A 92 Y L92 Y L92 Y 93 V H89 V H89 V 93 T L93 T L93 T 94 YH90 Y H90 Y 94 T L94 T L94 T 95 Y H91 Y H91 Y 95 P L95 P L95 P 96 C H92C H92 C 96 P L96 P L96 P 97 S H93 S H93 S 97 T L97 T L97 T 98 R H94 RH94 R 98 F L98 F L98 F 99 W H95 W H95 W 99 G L99 G L99 G 100 G H96 G H96G 100 Q L100 Q L100 Q 101 G H97 G H97 G 101 G L101 G L101 G 102 D H98 DH98 D 102 T L102 T L102 T 103 G H99 G H99 G 103 K L103 K L103 K 104 FH100 F H100 F 104 V L104 V L104 V 105 Y H100A Y H100A Y 105 E L105 EL105 E 106 A H100B A H100B A 106 I L106 I L106 I 107 M H100C M H100C M107 K L107 K L107 K 108 D H101 D H101 D 108 R 108 109 Y H102 Y H102 Y109 T 109 110 W H103 W H103 W 110 V 110 111 G H104 G H104 G 111 A 111112 Q H105 O H105 Q 112 A 112 113 G H106 G H106 G 113 P 113 114 T H107 TH107 T 114 S 114 115 L H108 L H108 L 115 V 115 116 V H109 V H109 V 116 F116 117 T H110 T H110 T 117 I 117 118 V H111 V H111 V 118 F 118 119 SH112 S H112 S 119 P 119 120 S H113 S H113 S 120 P 120 121 A 118 121 S121 122 S 119 122 D 122 123 T 120 123 E 123 124 K 121 124 Q 124 125 G122 125 L 125 126 P 123 126 K 126 127 S 124 127 S 127 128 V 125 128 G128 129 F 126 129 T 129 130 P 127 130 A 130 131 L 128 131 S 131 132 A129 132 V 132 133 P 130 133 V 133 134 S 131 134 C 134 135 S 132 135 L135 136 K 133 136 L 136 137 S 134 137 N 137 138 T 135 138 N 138 139 S136 139 F 139 140 G 137 140 Y 140 141 G 138 141 P 141 142 T 139 142 R142 143 A 140 143 E 143 144 A 141 144 A 144 145 L 142 145 K 145 146 G143 146 V 146 147 C 144 147 Q 147 148 L 145 148 W 148 149 V 146 149 K149 150 K 147 150 V 150 151 D 148 151 D 151 152 Y 149 152 N 152 153 F150 153 A 153 154 P 151 154 L 154 155 E 152 155 Q 155 156 P 153 156 S156 157 V 154 157 G 157 158 T 155 158 N 158 159 V 156 159 S 159 160 S157 160 Q 160 161 W 158 161 E 161 162 N 159 162 S 162 163 S 160 163 V163 164 G 161 164 T 164 165 A 162 165 E 165 166 L 163 166 Q 166 167 T164 167 D 167 168 S 165 168 S 168 169 G 166 169 K 169 170 V 167 170 D170 171 H 168 171 S 171 172 T 169 172 T 172 173 F 170 173 Y 173 174 P171 174 S 174 175 A 172 175 L 175 176 V 173 176 S 176 177 L 174 177 S177 178 Q 175 178 T 178 179 S 176 179 L 179 180 S 177 180 T 180 181 G178 181 L 181 182 L 179 182 S 182 183 Y 180 183 K 183 184 S 181 184 A184 185 L 182 185 D 185 186 S 183 186 Y 186 187 S 184 187 E 187 188 V185 188 K 188 189 V 186 189 H 189 190 T 187 190 K 190 191 V 188 191 V191 192 P 189 192 Y 192 193 S 190 193 A 193 194 S 191 194 C 194 195 S192 195 E 195 196 L 193 196 V 196 197 G 194 197 T 197 198 T 195 198 H198 199 Q 196 199 Q 199 200 T 197 200 G 200 201 Y 198 201 L 201 202 I199 202 S 202 203 C 200 203 S 203 204 N 201 204 P 204 205 V 202 205 V205 206 N 203 206 T 206 207 H 204 207 K 207 208 K 205 208 S 208 209 P206 209 F 209 210 S 207 210 N 210 211 N 208 211 R 211 212 T 209 212 G212 213 K 210 213 E 213 214 V 211 214 C 214 215 D 212 216 K 213 217 K214 218 V 215 219 E 216 220 P 217 221 K 218 222 S 219 223 C 220 224 D221 225 K 222 226 T 223 227 H 224 228 T 225 229 C 226 230 P 227 231 P228 232 C 229 233 P 230 234 A 231 235 P 232 236 E 233 237 L 234 238 L235 239 G 236 240 G 237 241 P 238 242 S 239 243 V 240 244 F 241 245 L242 246 F 243 247 P 244 248 P 245 249 K 246 250 P 247 251 K 248 252 D249 253 T 250 254 L 251 255 M 252 256 I 253 257 S 254 258 R 255 259 T256 260 P 257 261 E 258 262 V 259 263 T 260 264 C 261 265 V 262 266 V263 267 V 264 268 D 265 269 V 266 270 S 267 271 H 268 272 E 269 273 D270 274 P 271 275 E 272 276 V 273 277 K 274 278 F 275 279 N 276 280 W277 281 Y 278 282 V 279 283 D 280 284 G 281 285 V 282 286 E 283 287 V284 288 H 285 289 N 286 290 A 287 291 K 288 292 T 289 293 K 290 294 P291 295 R 292 296 E 293 297 E 294 298 Q 295 299 Y 296 300 N 297 301 S298 302 T 299 303 Y 300 304 R 301 305 V 302 306 V 303 307 S 304 308 V305 309 L 306 310 T 307 311 V 308 312 L 309 313 H 310 314 Q 311 315 D312 316 W 313 317 L 314 318 N 315 319 G 316 320 K 317 321 E 318 322 Y319 323 K 320 324 C 321 325 K 322 326 V 323 327 S 324 328 N 325 329 K326 330 A 327 331 L 328 332 P 329 333 A 330 334 P 331 335 I 332 336 E333 337 K 334 338 T 335 339 I 336 340 S 337 341 K 338 342 A 339 343 K340 344 G 341 345 Q 342 346 P 343 347 R 344 348 E 345 349 P 346 350 Q347 351 V 348 352 Y 349 353 T 350 354 L 351 355 P 352 356 P 353 357 S354 358 R 355 359 E 356 360 E 357 361 M 358 362 T 359 363 K 360 364 N361 365 Q 362 366 V 363 367 S 364 368 L 365 369 T 366 370 C 367 371 L368 372 V 369 373 K 370 374 G 371 375 F 372 376 Y 373 377 P 374 378 S375 379 D 376 380 I 377 381 A 378 382 V 379 383 E 380 384 W 381 385 E382 386 S 383 387 N 384 388 G 385 389 Q 386 390 P 387 391 E 388 392 N389 393 N 390 394 Y 391 395 K 392 396 T 393 397 T 394 398 P 395 399 P396 400 V 397 401 L 398 402 D 399 403 S 400 404 D 401 405 G 402 406 S403 407 F 404 408 F 405 409 L 406 410 Y 407 411 S 408 412 K 409 413 L410 414 T 411 415 V 412 416 D 413 417 K 414 418 S 415 419 R 416 420 W417 421 Q 418 422 Q 419 423 G 420 424 N 421 425 V 422 426 F 423 427 S424 428 C 425 429 S 426 430 V 427 431 M 428 432 H 429 433 E 430 434 A431 435 L 432 436 H 433 437 N 434 438 H 435 439 Y 436 440 T 437 441 Q438 442 K 439 443 S 440 444 L 441 445 S 442 446 L 443 447 S 444 448 P445 449 G 446

TABLE 2 Exemplary site-specific heavy chain modifications. The left-mostcolumn refers to the Kabat Chothia number (if less than 118) or the EUnumber (if 118 or greater.) The columns numbered 1-27 refer to SSHC-1 toSSHC-27, as recited in this disclosure. HC Residue No. 1 2 3 4 5 6 7 8 910 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27  3 x x x  5 x x xx x x x  7 x x x x  14 x x x x  16 x x x  19 x x x x x x x x x  23 x x xx x x x x x x x  25 x x x x x x x x x x  40 x x x x x x x x  42 x x x xx x x  43 x x x x  51 x x x  52 x x x x x x x  53 x x  56 x x  65 x x xx  70 x x x x x x x x  71 x  74 x x x x x x  77 x  79 x   82A x x x  84x x x x x x x x x x x  98 x x x 100 x x x x 110 x x x x x x x 112 x x xx x x 117 x 118 x x x x x x x x x x x 119 x x x x x x x x x x x x x x x121 x x x x x x x x x x 124 x 132 x x x x x x x 134 x x x x x x x 135 xx x x x x x x x x 136 x x x x x x x x x x x x x x x x 137 x x x x x x xx x x 138 x x x x 139 x x x x x x x 155 x x x x 160 x x x x x x x x x xx 161 x x x 162 x x x x x x x x x x x 164 x x 165 x x x x x x 172 x x xx x x x 174 x x x x 176 x x x x 177 x x x x 180 x x x x x x x x x x 183x 184 x 187 x x 190 x x x x x x x x 191 x x x x x x 192 x x x 193 x 194x x x x x x 195 x x x x 197 x x x 214 x x x x x x 216 x x x x x 219 x xx x x x x x 221 x x x x x x 222 x x x x x x x x x 224 x 225 x x x x 227x x x x 230 x x x x 231 x x x x 232 x x x x 236 x x x x 238 x x x x 239x x x x x x x 241 x x x x x x x x x x 243 x x x x 246 x x x x x x 260 x262 x x x x 264 x x x x 265 x x x x 267 x x x x x x x x x x x 268 x x xx x x x 269 x x x x x x x 270 x x x x x x x 271 x x x x x x x 272 x x xx x x x x x x 274 x x x x x x x x 275 x x x x x x 278 x x x x 280 x x xx x x 281 x x x x x x 282 x x x x x x x x x x x 283 x x x x x x 286 x xx x x x x 289 x x x x x x x 292 x x x x x x x x x 293 x x x x x x x x xx x x 294 x x x x x x x 295 x x x x x x x 296 x x x x x x x x x x x x297 x x x x x x x x x x x 298 x x x x x x x x x x x x 299 x x x x x x x300 x x x x x x x 301 x x x x x x x 303 x x x x x x x x x x x 305 x x xx x x x x x x x 317 x x x x x x 320 x x x x x x 324 x x x x x x 326 x xx x x x 327 x x x x x x 329 x x x x x x x 330 x x x x x x x x 332 x x xx x x x 333 x x x x x x x 334 x x x x x x x x x x x 335 x x x x x x x xx 337 x x x x x x 339 x x x x x x 340 x x x x x x x x x x x 341 x x x x342 x x x x x x x x x 343 x x 344 x x x x x x 355 x x x x x x x x x x xx 356 x x x x 358 x x x x 359 x x x x x x x 360 x x x x 361 x x 362 x x375 x x x x x x 383 x x x x 384 x x x x 386 x x x x x x x x 389 x x x xx x x 392 x x x x x x x x x x 398 x x x x x x 400 x 404 x x x x x x x xx x x x x 407 x 420 x x x x x x x 421 x x x x x x x 422 x x x 424 x x x436 x x x x 438 x x x x x x x x x x x 440 x x x 442 x x x x 443 x x x

TABLE 3 Exemplary site-specific light chain modifications. The left-mostcolumn refers to the Kabat or Chothia number (if less than 108) or theEU number (if 108 or greater). The columns numbered 1-22 to SSLC-1 toSSLC-27, as recited in this disclosure. LC Residue No. 1 2 3 4 5 6 7 8 910 11 12 13 14 15 16 17 18 19 20 21 22 −1 x x x x x x x x x x x x x 3 xx x x x 5 x x x x x x x 7 x x x x x x x x x x x x 8 x x x x x x x x x xx 9 x x x x x 10 x x x 14 x x 16 x x x x x x x x x x x x x 17 x x x x xx x 18 x x x x x x x 20 x x x x x x x 22 x x x x x x x x x x x x 26 x xx x x 27 x x x x x x x 43 x x x x x x 45 x x x x x x x 49 x x x x x x xx 56 x x x x x x x x 57 x x x x x x x x 58 x x x x x x x 60 x x x x x xx x 63 x x x x x x x x x x x x x 65 x x x x x 66 x x x x x 67 x x x x xx x x 68 x x x x x x 70 x x x x x x x x x 77 x x x x x x x 79 x x x x xx x 107 x x x x x x x 109 x x x x x x x x 112 x x x x 114 x x x 138 x xx x x x 142 x x x x x x x x x x x 143 x x x x x x x 144 x x x x x x 152x x x x x x x x x x x x 153 x x x x x x x x 156 x x x x x x 157 x x 168x x x x 171 x x x x x 182 x x x x x x x 184 x x x x x x 188 x x x x x xx 199 x x x x x x x x x 201 x x x x x 202 x x x x x x x x 203 x x x x xx x x 206 x x

1-11. (canceled)
 12. A polypeptide comprising at least one amino acid residues of the compound of Formula I:

or a salt thereof, wherein: Ar is

V is a single bond, lower alkylene, or —W₁—W₂—; one of W₁ and W₂ is absent or lower alkylene, and the other is —NH—, —O—, or —S—; X₁ is —NH—, —O—, or —S—; one of Z₁, Z₂, and Z₃ is —N— and the others of Z₁, Z₂, and Z₃ are each independently —CH—; and R is lower alkyl.
 13. The polypeptide of claim 12 comprising at least two amino acid residues of the compound of Formula I.
 14. The polypeptide of any of claim 12, further comprising at least one amino acid residue comprising an azide functional group.
 15. The polypeptide of claim 14, wherein each amino acid residue comprising an azide functional group is selected from the group consisting of a residue of a compound according to any one of formulas A1-A30, and A40:


16. A conjugate comprising the polypeptide of claim 12 linked to a payload, where the link is formed by a reaction of the tetrazole of the at least one amino acid residues, with a payload comprising a strained alkene functional group, and optionally comprising a linking moiety between the polypeptide and the payload.
 17. An antibody comprising at least one amino acid residue corresponding to the compound of Formula I:

or a salt thereof, wherein: Ar is

V is a single bond, lower alkylene, or —W₁—W₂—; one of W₁ and W₂ is absent or lower alkylene, and the other is —NH—, —O—, or —S—; X₁ is —NH—, —O—, or —S—; one of Z₁, Z₂, and Z₃ is —N— and the others of Z₁, Z₂, and Z₃ are each independently —CH—; and R is lower alkyl.
 18. The antibody of claim 17 comprising at least two amino acid residues of the compound of Formula I.
 19. The antibody of claim 17, further comprising at least one amino acid residue comprising an azide functional group.
 20. The antibody of claim 19, wherein each amino acid residue comprising an azide functional group is selected from the group consisting of a residue of a compound according to any one of formulas A1-A30, and A40:


21. A conjugate comprising the antibody of claim 17 linked to a payload, where the link is formed by a reaction of the tetrazole of the at least one amino acid residues, with a payload comprising a strained alkene functional group, and optionally comprising a linking moiety between the antibody and the payload.
 22. An orthogonal tRNA aminoacylated with an amino acid residue corresponding to the compound of Formula I:

or a salt thereof, wherein: Ar is

V is a single bond, lower alkylene, or —W₁—W₂—; one of W₁ and W₂ is absent or lower alkylene, and the other is —NH—, —O—, or —S—; X₁ is —NH—, —O—, or —S—; one of Z₁, Z₂, and Z₃ is —N— and the others of Z₁, Z₂, and Z₃ are each independently —CH—; and R is lower alkyl.
 23. A method of producing a polypeptide, comprising contacting a first polypeptide with the orthogonal tRNA of claim 22 under conditions suitable for incorporating the amino acid residue into the first polypeptide.
 24. The method of claim 23, wherein the orthogonal tRNA base pairs with a codon that is not normally associated with an amino acid.
 25. The method of claim 23, wherein the contacting occurs in a reaction mixture which comprises a tRNA synthetase that acylates the orthogonal tRNA with the compound of Formula I.
 26. The method of claim 25, wherein the tRNA synthetase is selected from the group consisting of SEQ ID NOs: 4-5.
 27. The conjugate of claim 16, wherein V in the compound of Formula I is a single bond, —NH—, or —CH₂NH—.
 28. The conjugate of claim 16, wherein the compound according to Formula I is according to formula IV, V, VI or VII:

or a salt thereof, wherein V is a single bond, —NH—, or —CH₂NH—.
 29. The conjugate of claim 16, wherein the compound according to Formula I is according to any one of formulas 2, 3, and 6-10:

or a salt thereof.
 30. The antibody of claim 17, wherein V in the compound of Formula I is a single bond, —NH—, or —CH₂NH—.
 31. The antibody of claim 17, wherein the compound according to Formula I is according to formula IV, V, VI or VII:

or a salt thereof, wherein V is a single bond, —NH—, or —CH₂NH—.
 32. The antibody of claim 17, wherein the compound according to Formula I is according to any one of formulas 2, 3, and 6-10:

or a salt thereof.
 33. The conjugate of claim 21, wherein V in the compound of Formula I is a single bond, —NH—, or —CH₂NH—.
 34. The conjugate of claim 21, wherein the compound according to Formula I is according to formula IV, V, VI or VII:

or a salt thereof, wherein V is a single bond, —NH—, or —CH₂NH—.
 35. The conjugate of claim 21, wherein the compound according to Formula I is according to any one of formulas 2, 3, and 6-10:

or a salt thereof.
 36. The conjugate of claim 21, wherein the linking moiety comprises a peptide linkage.
 37. The conjugate of claim 21, wherein the linking moiety comprises PEG.
 38. The conjugate of claim 21, wherein the payload is a label or a cytotoxic compound, or a combination thereof. 